蒙古冰草MwLEA3基因功能研究及MwRRT基因的克隆
|
摘要
蒙古冰草(Agropyron mongolicum Keng)是禾本科冰草属小麦族多年生草本植物,不仅具有极高的饲用价值,而且具有很强的抗逆性,富含大量的抗旱、抗寒、耐盐基因,可以为牧草及近缘种作物(水稻、小麦、玉米等)的抗性改良及新品种选育提供宝贵的资源。本研究分离出蒙古冰草MwLEA3基因并进行了功能验证,并首次克隆得到了蒙古冰草类反转录转座子。主要研究结果如下:
1.分离了蒙古冰草MwLEA3基因,其全长为705bp,开放阅读框为492bp,编码163个氨基酸。
2.构建了蒙古冰草MwLEA3基因的亚细胞定位融合蛋白载体pA7-MwLEA3,并将此载体及pA7-GFP载体质粒转化至洋葱细胞中进行亚细胞定位。试验结果表明,pA7-MwLEA3融合蛋白集中分布在细胞核中,而对照pA7-GFP在整个细胞中均有分布,证明了MwLEA3基因表达的蛋白是核定位蛋白。
3.成功构建蒙古冰草MwLEA3基因植物表达载体pCAM-MwLEA3,并经农杆菌介导进行烟草的遗传转化。对经过抗性检测的转基因烟草T_0代植株进行PCR检测、PCR-Southern、RT-PCR鉴定以及干旱胁迫试验。分子检测结果表明,MwLEA3基因已整合到烟草基因组中,并在转录水平正常表达;干旱胁迫试验结果显示转基因烟草植株的抗旱性高于野生型烟草植株。
4.对10个株系转基因烟草T_1代的卡那霉素抗性进行研究表明,10个株系中,有7个株系的T_1代的分离比例为3:l,符合孟德尔式遗传规律,这表明MwLEA3基因是单拷贝插入;另外3个株系的T_1代不符合孟德尔式3:1的遗传规律,说明这些植株中的MwLEA3基因可能是多个拷贝插入的。
5.构建了35S启动子调控的具有MwLEA3基因反向重复结构的ihpRNA表达载体pART27-MwLEA3(HB)-MwLEA3(EX),并通过冻融法转化将MwLEA3基因的ihpRNA表达框架成功导入根癌农杆菌LBA4404中。利用带有目的表达载体的农杆菌菌液侵染蒙古冰草愈伤组织,现正在筛选分化培养基上培养,后续试验正在进行中。
6.首次克隆得到了蒙古冰草类反转录转座子(MwRRT)基因,该基因片段长度为1005bp,在1～225bp为非编码区,长度为225bp,其氨基酸序列在226～1005bp,共780bp,编码260个氨基酸。核苷酸及氨基酸同源性对比分析发现,所克隆基因与小麦(Triticum aestivum)、水稻(Oryza sativa)、大麦(Hordeum vulgare)、高粱(Sorghum bicolor)的LTR类反转录转座子的核苷酸同源性约70%,氨基酸同源性约50%,GenBank登陆号NO.GQ855806.1。
Mongolia Wheatgrass (Agropyron mongolicum Keng), a perennial grass, belongs to Agropyron genus in Gramineae, not only has high feeding value, but also rich in resistant genes,such as drought-tolerant gene, cold- tolerant gene, salt-tolerant gene and so on. It could provide valuable resource for the improvement of resistance and breeding of forage and its closely related species including rice, wheat, maize and so on. In this paper, the function of the MwLEA3 gene of Mongolia Wheatgrass was verified on the basis of molecular biological identification and physiological and biochemical identification,and MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. The main results are as follows:
1. The full length MwLEA3 gene of Mongolia Wheatgrass was cloned by RT-PCR amplification technique. DNAman analysis indicated its open read frame was 492bp, coded 163 amino acids.
2. The fusion protein vector pA7-MwLEA3 for subcellular localization was constructed. The fusion protein vector and pA7-GFP vector plasmids were transformed into onion cells. The results showed that the fusion protein pA7-MwLEA3 was mainly distributed in the cell nucleus, while pA7-GFP were distributed in the whole cell. This result was demonstrating that the expressed protein encoded by MwLEA3 was a nuclear localized protein.
3. The plant expression vector of pCAM-MwLEA3 was constructed and transformed into tobacco via Agrobacterium infestation. T_0 transformed tobaccos were tested by PCR、PCR-Southern、RT-PCR and drought stress. Molecular detecting results showed that MwLEA3 gene had been transformed into tobacco and expressed. The results of drought stress showed that the transgenic tabaccos had stronger resistance than wide-type ones.
4. The results of kanamycin resistant study of 10 lines T_1 generation of transgenic tobacco plants showed that the separation law of T_1 generation of seven lines of transgenic tobacco was 3:1, showing coincidence with Mendelian genetic law. Other three lines did not show seperation ratio of 3:1.
5. The ihpRNA expression vector was constructed with inverted repeats of MwLEA3 gene, which was promoted by 35S promoter. The voctor was transferred into Agrobacterium tumefaciens strain LBA4404 with freeze-thaw method. The callus of Mongolia Wheatgrass were infected with Agrobacterium tumfaciens containing the ihpRNA expression vector, and are now being cultured on differentiation medium.
6. MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. It had a length of 1005bp, and consisted of a non-coding region from 1bp to 225bp and a coding region from 226bp to 1005bp that encoded 260 amino acids. Sequence comparison analysis with LTR class retrotransposons from wheat (Triticum aestivum), rice (Oryza sativa), barley (Hordeum vulgare) and sorghum (Sorghum bicolor) showed that it had about 70% idendity in nucleotide sequence and about 50% homology in amino acid sequence. We registered this gene in GenBank and the accession No. was GQ855806.1.
1.分离了蒙古冰草MwLEA3基因,其全长为705bp,开放阅读框为492bp,编码163个氨基酸。
2.构建了蒙古冰草MwLEA3基因的亚细胞定位融合蛋白载体pA7-MwLEA3,并将此载体及pA7-GFP载体质粒转化至洋葱细胞中进行亚细胞定位。试验结果表明,pA7-MwLEA3融合蛋白集中分布在细胞核中,而对照pA7-GFP在整个细胞中均有分布,证明了MwLEA3基因表达的蛋白是核定位蛋白。
3.成功构建蒙古冰草MwLEA3基因植物表达载体pCAM-MwLEA3,并经农杆菌介导进行烟草的遗传转化。对经过抗性检测的转基因烟草T_0代植株进行PCR检测、PCR-Southern、RT-PCR鉴定以及干旱胁迫试验。分子检测结果表明,MwLEA3基因已整合到烟草基因组中,并在转录水平正常表达;干旱胁迫试验结果显示转基因烟草植株的抗旱性高于野生型烟草植株。
4.对10个株系转基因烟草T_1代的卡那霉素抗性进行研究表明,10个株系中,有7个株系的T_1代的分离比例为3:l,符合孟德尔式遗传规律,这表明MwLEA3基因是单拷贝插入;另外3个株系的T_1代不符合孟德尔式3:1的遗传规律,说明这些植株中的MwLEA3基因可能是多个拷贝插入的。
5.构建了35S启动子调控的具有MwLEA3基因反向重复结构的ihpRNA表达载体pART27-MwLEA3(HB)-MwLEA3(EX),并通过冻融法转化将MwLEA3基因的ihpRNA表达框架成功导入根癌农杆菌LBA4404中。利用带有目的表达载体的农杆菌菌液侵染蒙古冰草愈伤组织,现正在筛选分化培养基上培养,后续试验正在进行中。
6.首次克隆得到了蒙古冰草类反转录转座子(MwRRT)基因,该基因片段长度为1005bp,在1～225bp为非编码区,长度为225bp,其氨基酸序列在226～1005bp,共780bp,编码260个氨基酸。核苷酸及氨基酸同源性对比分析发现,所克隆基因与小麦(Triticum aestivum)、水稻(Oryza sativa)、大麦(Hordeum vulgare)、高粱(Sorghum bicolor)的LTR类反转录转座子的核苷酸同源性约70%,氨基酸同源性约50%,GenBank登陆号NO.GQ855806.1。
Mongolia Wheatgrass (Agropyron mongolicum Keng), a perennial grass, belongs to Agropyron genus in Gramineae, not only has high feeding value, but also rich in resistant genes,such as drought-tolerant gene, cold- tolerant gene, salt-tolerant gene and so on. It could provide valuable resource for the improvement of resistance and breeding of forage and its closely related species including rice, wheat, maize and so on. In this paper, the function of the MwLEA3 gene of Mongolia Wheatgrass was verified on the basis of molecular biological identification and physiological and biochemical identification,and MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. The main results are as follows:
1. The full length MwLEA3 gene of Mongolia Wheatgrass was cloned by RT-PCR amplification technique. DNAman analysis indicated its open read frame was 492bp, coded 163 amino acids.
2. The fusion protein vector pA7-MwLEA3 for subcellular localization was constructed. The fusion protein vector and pA7-GFP vector plasmids were transformed into onion cells. The results showed that the fusion protein pA7-MwLEA3 was mainly distributed in the cell nucleus, while pA7-GFP were distributed in the whole cell. This result was demonstrating that the expressed protein encoded by MwLEA3 was a nuclear localized protein.
3. The plant expression vector of pCAM-MwLEA3 was constructed and transformed into tobacco via Agrobacterium infestation. T_0 transformed tobaccos were tested by PCR、PCR-Southern、RT-PCR and drought stress. Molecular detecting results showed that MwLEA3 gene had been transformed into tobacco and expressed. The results of drought stress showed that the transgenic tabaccos had stronger resistance than wide-type ones.
4. The results of kanamycin resistant study of 10 lines T_1 generation of transgenic tobacco plants showed that the separation law of T_1 generation of seven lines of transgenic tobacco was 3:1, showing coincidence with Mendelian genetic law. Other three lines did not show seperation ratio of 3:1.
5. The ihpRNA expression vector was constructed with inverted repeats of MwLEA3 gene, which was promoted by 35S promoter. The voctor was transferred into Agrobacterium tumefaciens strain LBA4404 with freeze-thaw method. The callus of Mongolia Wheatgrass were infected with Agrobacterium tumfaciens containing the ihpRNA expression vector, and are now being cultured on differentiation medium.
6. MwRRT retrotransposons was cloned from Mongolia Wheatgrass for the first time. It had a length of 1005bp, and consisted of a non-coding region from 1bp to 225bp and a coding region from 226bp to 1005bp that encoded 260 amino acids. Sequence comparison analysis with LTR class retrotransposons from wheat (Triticum aestivum), rice (Oryza sativa), barley (Hordeum vulgare) and sorghum (Sorghum bicolor) showed that it had about 70% idendity in nucleotide sequence and about 50% homology in amino acid sequence. We registered this gene in GenBank and the accession No. was GQ855806.1.
引文
1崔清涛,刘清泉,阚丽梅.蒙古冰草(Agropyron mongolicun)生物量季节动态的研究[J].内蒙古林业科技,1994,3:51-53
2李志勇,陈凤林,陈立波,等.优良禾草新品种—蒙古冰草[J].中国草地,1993,1:74,78
3李立会,扬新民.通过属间杂交向小麦转移冰草基因的研究[J].中国农业科学, 1998,31(6):1-5
4高卫华,云锦凤,杨静等.冰草属牧草营养器官解剖学研究[J].内蒙古草业,1990,4:19-22
5王怡丹,郭晓宇,全炳武.水分胁迫下蒙古冰草、扁穗冰草和滨麦抗旱性研究[J].延边大学农学学报,2008,30(2):98-104
6王荣华,石雷,汤庚国,等.渗透胁迫对蒙古冰草幼苗保护酶系统的影响[J].植物学通报,2003, 20(3):330-335
7阎贵兴,张素贞.沙芦草染色体组型的分析.中国草地学报,1985,2:38-40
8段晓刚,王丽,卜秀玲.蒙古冰草和布顿大麦染色体组型分析.中国草地,1990(3):45-48
9 Chen Q, Jahidr J,Cauderon Y. Cytological studies on Agropyron Gaertn Species from Inner Mongolia,China[J].C.R.Acad.Sci.SerieⅢ,1989,309(11):519-525
10李立会,徐世雨.冰草属中B染色体在减数分裂期的遗传行为研究[J].植物学报,1991,33(11):833-839
11云锦凤,斯琴高娃.蒙古冰草B染色体的研究[J].内蒙古农牧学院学报,1996,17(1):14-17
12解新明.蒙古冰草(Agropyron mongolicum Keng)遗传多样性研究[D].内蒙古农业大学,2001
13解新明,云锦凤,赵冰,等.蒙古冰草遗传多样性的等位酶分析[J].草业科学,2001,6:6-11
14兰宝祥.蒙古冰草居群遗传多样性研究[J].中国农业科学,2005,38(3):468-473
15云锦凤,李瑞芬.冰草的远缘杂交及杂种分析[J].草地学报,1997,4:221-227
16乌仁其木格,于卓.几种小麦族禾草及其杂种酯酶同工酶研究[J].内蒙古农业大学学报(自然科学版),1999,4:49-53
17王月华,于卓,王四清,等.蒙古冰草×航道冰草杂种F3代的育性及染色体构型分析[J].中国草地,2003,25(4):1-7
18权威,于卓,王月华,等.蒙古冰草×航道冰草杂种F4代株系同工酶分析[J].草地学报,2006,14(1):14-19
19于卓,马艳红,李小雷,等.蒙古冰草与航道冰草正、反交F1及其染色体加倍植株AFLP分析[J].中国草地学报,2009,31(6):14-19
20赵彦,云锦凤.蒙古冰草Lea3抗旱基因片段的分离与鉴定[J].华北农学报,2008,23(6):64-67
21康虹丽.蒙古冰草磷脂酶D基因全长cDNA序列的克隆及生物信息学分析[D].内蒙古农业大学,2008
22何军贤,傅家瑞.种子Lea蛋白的研究进展[J].植物生理学通讯,1996,32(9):291-246
23 Dure L,Greenway SC,Galau GA. Developmental biochemistry of cotton seed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by invitro and invivo protein synthesis[J].Biochemistry,1981,20(14): 4162-4168
24 Grzelczak ZF,Sattolo MH,Hanleybowdoin LK, et al., Synthesis and turnover of proteins and messenger-RNA in germinating wheat embryos[J].Canadian Journal of Biochemistry,1982,60:389-397
25 Bartels D, Singn M, Salamini F. Onset of desiccation tolerance during development of the barley embryo[J]. Planta,1988,175(4): 485-492
26 Bostock RM,Quatrano RS.Regulation of em gene-expression in Rice:Interaction between osmotic-stress and abscisic acid[J].Plant Physiology,1992,98(4): 1356- 1363
27 Campbell SA,Crone DE,Ceccardi TL,et al.A ca.40 kDa maize (Zea mays L.)embryo dehydrin is encoded by the dhn2 locus on chromosome 9[J].Plant Molecula Biology,1998,38(3):417-423
28 Hsing YC,Chen ZY,Shih MD,et al.Unusual sequences of group 3 LEA mRNA inducible by maturation or drying in soybean seeds[J].Plant Mol Biol,1995,29(4):863-868
29 BRAY EA.Molecular responses to water deficit[J].Plant Physiol,1993,103:1035-1040
30 Franz G, Hatzopoulos P,Jones TJ,et al. Molecular and genetic analysis of an embryonic gene, DC 8, from Daucus carota L. Mol Gen Genet, 1989, 218(1): 143-151
31 Prieto DP,Almoguera C,Rojas A, et al. Seed-specific expression patterns and regulation by ABI3 of an unusual late embryogenesis-abundant gene in sunflower[J]. Plant Mol Biol, 1999,39(3):615-627
32 Ingram J,Bartels D.The molecular basis of dehydration tolerance in plants[J].Annu Rev Plant Physiol Plant Mol Biol,1996,47:377-403
33 Hughes DW,Galau GA.Developmental and environmental induction of LEA and LEAA messenger-RNAS and the postabscission program during embryo culture[J].Plant Cell,1991,3(6):605-618
34 Caray Arroyo A,Colmenero Flores JM,Garoiarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit[J].Biological Chemistry,2000,275(8):5668-5674
35 Wise MJ, Tunnacliffe A.POPP the question:what do LEA proteins do?[J].Trends Plant Science,2004,9(1):13-17
36 Hollung K, Espelund M, Jakobsen KS. Another LEA B19 gene (Group1 LEA) from barley containing a single 20 amino acid hydrophilic motif[J]. Plant Molecular Biology, 1994,25(3):559-564
37 Swire Clark GA, Marcotle WR. The wheat LEA protein Em functions as an osmoprotective molecule in Sacharomyces cerevisiae. Plant Biology,1999,39(1):117-128
38 Stacy RAP, Espelund M, Saeboe-Larssen S,et al. Evolution of the Group-1 Late Embryogenesis abundant(LEA) genes analysis of the LEA B19 gene family in barley[J]. Plant Molecular Biology,1995,28(6):1039-1054
39 Bray EA, Alterations in Gene Expression in Response to Water Deficit. In: Basra AS Stress-induced Gene Expression in Plants,Newark:Harwood Academic, 1994, 1-23
40 Koag MC,Wilkens S,Fenton RD,et al. The K-segment of maize DHN1 mediates binding to anionic phospholipid vesicles and concomitant structural changes[J].Plant Physiology,2009,150(3):1503-1514
41宋松泉,陈玲,傅家瑞.种子脱水耐性与LEA蛋白[J].植物生理学通讯,1999,35(5):424-431
42 Rorat T.Plant dehydrins-tissue location, structure and function[J].Cell.Mol. Biol.Lett.,2006,11(4):536-556
43 Danyluk J, Perron A, Houde M,et al. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat[J].Plant cell,1988,10(4):623-638
44 Close TJ. Dehydrins: A commonality in the response of plants to dehydration and low temperature[J].Physiolgia Plantarum.1997,100(2):291-296
45 Curry J, Walter-Simmons MK. Unusual sequence of group 3 LEA(Ⅱ) mRNA inducible by dehydration stress in wheat[J].Plant Molecular Biology,1993,21(5),907-912
46 Straub PF, Shen QX, Ho T-HD. Structure and promoter analysis of an ABA and stress regulated barley gene, HVA1 [J].Plant Mol. Biol., 1994,26(2):617-630
47 Dure L.A repeating 11-mer amino acid motif and plant desiccation[J].Plant Journal,1993,3(3):363-369
48孙海丹,兰英,刘昀,等.LEA蛋白质11-氨基酸基序与植物抗旱性[J].东北师范大学学报:自然科学版,2004,36(3):85-90
49 Kim HS,Lee JH,Kim JJ, et al.Molecular and functional characterization of CaLEA6, the gene for a hydropobic LEA protein from Capsicum annuum[J]. Gene,2005,344: 115-123
50 Maitra N,Cushman JC.Isolation and characterzation of a drought-induced soybean cDNA-encoding a D95 family late-embyogenesis-abundant protein[J].Plant Physiology,1994,106(2):805-806
51 Roberts JK,DeSimone NA.Cellular concentrations and unifomuty of cell-type accumulation of two Lea proteins in cotton embryos.Plant Cell,1993,5: 769-780
52 Goday A,Jensen AB,Culianez-Macia FA,et al.The maize abscisic acid-responsiveprotein Rab 17 is located in the nucleus Cell and interacts with nuclear localization signals[J]. Plant Cell,1994, 6:351-360
53 Asghar R,Fenton RD,DeMason DA,et al.Nuclear and cytoplasmic localization of maize embryo and aleurone dehydrin[J].Protoplasma,1994,171:87-94
54 Close TJ.Dehydrins:Emergence of a biochemical role of a family of plant dehydration protein[J].Physiol Plant,1996,97:795-803
55 Ukaji N,Kuwabara C,Takezawa D,et al.Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3 late-embryogenesis abundant proteins[J].Plant Physiology,2001,12(4)6: 1588-1597
56 Jensen AB, Goday A, Figueras M, et al. Phosphorylation mediates the nuclear targeting of the maize Rab17 protein[J].Plant Journal,1998,13(5):691-697
57 Alsheikh MK,Heyen BJ, Randall SK. Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation [J]. Journal of Biological Chemistry,2003,278(42): 40882-40889
58 Nair R,Rost B. LOCnet and LOCtarget:sub-cellular localization for structural genomics targets[J].Nucleic Acids Research,2004,32:517-521
59 Xu DP,Duan XL,Wang BY,et al.Expression of a late embryogenesis abundant protein gene,HVA1,from barley confers tolerance to water deficit and salt stress in transgenic rice [J].Plant Physiol,1996,110(1):249-257
60 Imai R,Chang L,Ohta A,et al.A lea-class gene of tomato congers salt and freezing tolerance when expressed inSaccharomyces cerevisiae[J].Gene,1996,170:243-248
61俞嘉宁,张林生,张劲松.小麦耐逆基因TaLEA3的克隆及在酵母中的功能分析[J].生物工程学报,2004,26(6),832-838
62赵咏梅,杨建雄,俞嘉宁,等.小麦耐逆基因-TaLEA2转化拟南芥的研究[J].西北植物学报,2006,26(1):1-6
63 Garay-Arroyo A,Colmenero-Flores JM,Garciarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit [J].J Biol Chem,2000,275:5668-5674
64袁克华,冯仁军,程萍,等.RNA干扰的研究进展[J].安徽农业科学,2009,37(8):3471-3473
65 Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811
66刘敏,曹毅,将彦.利用RNAi技术大规模分析基因功能的研究.植物学通报.2002.19(4):491-495
67 Cogoni C,Irelan JT, Schumacher M,et al.Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does notdepend on DNA-DNA interactions or DNA methylation. Embo journal,1996,15(12): 3153-3163
68朱旭东,黄培堂.RNA干涉的研究进展[J].生物技术通讯,2001,12(4):61-65
69石燕.RNAi实验技术研究进展[J].标记免疫分析与临床,2003,10(4):235-238
70 NyKanen A,Haley B,Zamore P D. ATP requirements and small interfering RNA structure in the RNA interference pathway[J].Cell,2001,107(3):309-321
71 Carmell MA.Germline transmission of RNAi in mice[J].Nature Struct.Biol.2003, 10:91-92
72 English JJ,Davenport GF,et al.Requirement of sense transcription for homology dependent virus resistance and trans-inactivation. Plant J,1996,12:597-603
73韩蓓,王秀敏,顾学范.双链RNA干涉技术(RNAi)在不同生物中应用的研究进展[J].遗传,2002,24(2):200-202
74 Wesley SV,Helliwell CA,Smith NA,et al.Construct design for efficient,effective and high-throughput gene silencing in plants.Plant J.200l,27(6):581-590
75朱俊华,竺晓平,温孚江,等.马铃薯Y病毒衣壳蛋白基因片段长度对RNA介导抗病性影响[J].中国科学C辑:生命科学,2004,34(1):23-30
76 Elbashir SM, Martinez J. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J].Embo journal,2001,20(23):6877-6888
77 Schweizer P,Pokorny J,Schulze-Lefert P,et al.Double-stranded RNA interferes with gene function at the single-cell level in cereals[J].Plant Journal,2000,24: 895-903
78 Liu Q,Singh SP,Green AG.High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing[J].Plant Physiology, 2002,129(4):1732-1743
79 Chuang CH,Meyerowitz EM.Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana[J].Proc.Natl.Acad. Sci.USA,2002,97: 4985-4990
80 Chi JT,Chang HY,Wang NN,et al. Genomewide view of gene silencing by small interfering RNAs[J].Proc.Natl.Acad.Sci.USA,2003,100(11):6343-6346
81周小云,陈信波,向建华. RNAi技术及在植物基因功能研究中的应用[J].生物学杂志,2005, 22(2):38-40,9
82徐化学,熊建华,傅彬英.应用Gateway技术构建水稻OsDAD1基因的RNA干涉载体[J].分子植物育种,2007,5(1):133-136
83 GATEWAY-(TM)——一种新型克隆技术[J].微生物学通报,2000,27(5):388
84 Curtis MD , Grossniklaus, U. gateway cloning vector set for high-throughput functional analysis of genes in planta[J].Plant physiology.2003,133(2):462-469
85谈心,杨宏,乔定君,等.干扰烟草GA 20-氧化酶siRNA植物表达载体的构建及矮化烟草的产生[J].应用与环境生物学报,2008,14(1):48-52
86柴晓杰,王丕武,关淑艳,等.应用RNA干扰技术降低玉米支链淀粉含量[J].植物生理与分子生物学学报,2005,31(6):625-630
87陈坚,兰涛,段远霖,等.水稻中SUSIBA2相似基因RNA干扰体系的建立[J].福建农林大学学报(自然科学版),2009,38(1):39-43
88彭昊,翟英,张芊,等.水稻高效RNA干涉体系的建立及其功能分析[J].中国农业科学,2006, 39(9):1729-1735
89刘玉汇.Patatin启动子驱动的马铃薯GBSSⅠ基因ihpRNAi植物表达载体的构建及转化马铃薯的研究[D].甘肃农业大学,2008
90李丽文,朱延明,李杰,等. RNAi技术在植物功能基因组学中的研究进展[J].东北农业大学学报,2007,38(1):119-124
91 Grimsley N, Hohn T, Boulton M L,et al. Agroinfection of Zea mays with maize streak virus DNA[J].Life sciences,1987,140:459-468
92 Ishida Y,Saito H. High efieiency transformation of maize(zea mays L.)mediated by Agrobacterium tumefaeiens[J].Natural Bioteehnol,1996,14(6):745-750
93 Hiei Y,Ohta S,Komari T.Effieient transformation of rice(Oryza sativa L.) mediated by Agrobaeterium and sequence analysis of the boundaries of the T-DNA[J].Plant Journal,1994,6:271-282
94 Sung HP, Shannon RM,Smith RH.T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices[J].Plant Molecular Biology,1996, 32(6):1135-1148
95 Cheng M,Fry JE,Pang SZ,et al. Genetic transformation of wheat mediated by Agro- bacterium tumefaciens[J].Plant Physiol,1997,115:971-980
96夏光敏,李忠谊. Transgeni根癌农杆菌介导的小麦转基因植株再生[J].植物生理学报, 1999,25(1):22-28
97胡张华,吴关庭,金卫.农杆菌介导的水稻转化及bar基因稳定遗传[J].浙江农业学报,2003,15(6):327-331
98肖军,石太渊,郑秀春,等.根癌农杆菌介导的高粱遗传转化体系的建立[J].杂粮作物,2004,24(4):200-203
99佘建明,张保龙,何晓兰,等.草地早熟禾农杆菌介导法基因转化条件[J].草地学报,2005,13(1): 39-43
100李达旭,张杰,赵建,等.根癌农杆菌介导转化川草二号老芒麦胚性愈伤组织[J].植物生理与分子生物学学报,2006,32 (1): 45-51
101胡繁荣.根癌农杆菌介导的金边狗牙根遗传转化条件的优化[J].植物资源与环境学报,2005,14 (2): 15-18
102张振霞,刘萍,杜雪玲,等.农杆菌介导GUS基因对多年生黑麦草转化的研究[J].广西植物,2007,27(1):121-126
103李进,侯海军,黄复深,等.基因枪介导获得转植酸酶基因水稻研究[J].2008,14(1):6-10
104李国圣,杨爱芳,张举仁,等.玉米愈伤组织的遗传转化及抗除草剂植株再生[J].科学通报, 2000,20:2181-2184
105魏松红,张领兵,张艳贞,等.小麦基因枪高效转化体系的建立[J].吉林农业科学,2001,2(3):7-11
106李志亮,黄丛林,王刚,等.利用基因枪法向高羊茅导入bar基因的研究[J].河北师范大学学报(自然科学版),2006,2:217-221
107 Zhou G Y,Weng J,Zeng Y, et al.Introduction of exogenous DNA into cotton embryos[J].Meth. Enzymo.,1983,101:433-481
108黄国存,董越梅,孙敬三,等.以GFP为标记用花粉管通道途径导入外源DNA[J].科学通报, 1998(23): 2531-2533
109武小霞,刘伟婷,刘琦,等.利用花粉管通道法将抗虫基因(cry V)转入大豆的研究[J].2010,29(4):565-568,574
110王秀君,郎志宏,陆伟,等.利用花粉管通道法将耐草甘膦基因mG2-epsps导入玉米自交系的研究初报[J].中国农业科技导报, 2008, 10(4):56-62
111沈世华,张秀君,郭奕明,等.玉米基因转化的离体子房注射及其转基因植株的鉴定[J].植物学报, 2001, 43: 1055-1057
112 Krens FA,Molendijk L,Wullems GJ,et al.In vitro transformation of plant protoplasts with Ti-plasmid[J].Nature,1982,296:72-74
113薛红卫,卫志明,许智宏.通过PEG法转化甘蓝获得转基因植株[J].植物学报,1997,39(1):28-33
114钟玉海,申艮宝.植物基因的遗传转化方法[J].安徽农学通报,2010,16(11):65-66,206
115任永霞,季静.植物遗传转化方法概述[J].河北北方学院学报(自然科学版),2005,21(6):38-42
116 Joersbo M,Brunstedt J.Directgene transfer to plantprotoplasts bymild sonication[J].Plant Cell Reports,1990,(9):207-210
117王关林,方宏筠.植物基因工程[M].科学出版社,2009,7
118余迪求,邓庆丽,沈亚楠,等.金鱼草基因转化和转基因植株再生[J].热带亚热带植物学报,1996,4(4):86-90
119贺竹梅.人促红细胞生成素基因在番茄中的表达[J].遗传学报,1998,25(2):155-159
120许新萍,陈金婷,张建中,等.抗稻瘟病和纹枯病的转基因水稻新品系[J].中山大学学报(自然科学版),2001,40(3):131-132
121 Dewal Jani, Laxman Singh Meena. Expression of cholera toxin B subunit in transgenic tomatoplant[J].Transgenic Research, 2002,11:447-454
122张荃,王淑芳,赵彦修,等.HAL1基因转化番茄及耐盐转基因番茄的鉴定[J].生物工程学报,2001,17(6):658-662
123 Samia Djennane, Jean-Eric Chauvin, Isabelle Quillere, et al. Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers[J].Transgenic Research,2002, 11:175-184
124王莉江,明小天,安成才,等.籼稻明恢63成熟种子愈伤组织的诱导及转基因水稻的抗性检测[J].生物工程学报,2002,18(3):323-327
125吴刚,崔海瑞,舒庆尧,等.转基因水稻中转录水平cry1Ab基因的沉默及其阶段复活[J].中国科学(C辑),2001,31(6):487-496
126曾千春,吴茜,周开达,等.修饰的cry1Ac基因导入籼稻明恢81获得抗虫纯合系[J].遗传学报,2002,29(6):519-524
127 Geng Sa,Ma Mi,Ye He-Chun, et al. Anther-specific expression ofiptgene in transgenic tobacco and its effect on plant development [J]. Transgenic Research,2002, 11: 269-278
128马媛媛.抗旱基因LEA的克隆及其序列解析[D].东北林业大学,2003
128邢浩然,刘丽娟,刘国振.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21:1-6
129 Abelson J, Simon M. Green fluorescent protein[J].Methods in Enzymology,1999,302: 449
130陈建勋,王晓峰.植物生理学实验指导[M].广州:华南理工大学出版社,2002
131高俊凤.植物生理学实验技术[M].世界图书出版社,2000
132 Bates LE,Waldren RP,Teare ID.Rapid determination of free proline for water stress studies[J].Plant Soil,1973,39:205-207
133李合生,孙群,赵世杰,等.植物生理生化实验原理和技术[M].北京:高等教育山出版社, 2000:260
134胡张华,吴关庭,金卫.农杆菌介导的水稻转化及bar基因稳定遗传[J].浙江农业学报,2003,15(6):327-331
135巩健,杨芳.单子叶植物表达载体的构建及农杆菌介导的玉米遗传转化的研究[J].生物技术,2007,17(3):2-5
136梁雪莲,孙毅.农杆菌介导转化小麦幼胚获得抗除草剂再生植株[J].植物生理与分子生物学学报,2003, 29(6): 501-506
137易自力,曹守云,王力,等.提高农杆菌转化水稻频率的研究[J].遗传学报,2001,28(4):352-358
138刘巧泉,张景六,王宗阳.根癌农杆菌介导的水稻高效转化系统的建立[J].植物生理学报,1999,24:259-271
139 Sahi S V,Chilton M D,Chilton W S.Corn metabolites affect growth and virulence of Agrobacterium tumefaciens[J]. Proc Natl Acad Sci USA.1990,27:3879-3883
140 Grandbastien M A. Retroelements in higher-plants[J].Trends Genet.,1992,8:103-108
141 Kunze R, Saedle H, Lonnig WE. Plant transposable elements[J].Adv.Bot.Res.,1997,27: 331-470
142 Voytas DF,Cummings MP,Konerczny AK,et al. Copia-like retrotransposons are ubiquity-ous among plants[J].Proc NAtl Acid Sci USA,1992,89(15):7124-7128
143 Kumar A,Bennetzen J I.Plant retrotransposons[J].Annu Rev Genet,1999,33:479-532
144刘柱,朱建清,赵建,等.植物反转录转座子的研究进展[J].生物化学与生物物理,2002,29 (4):527-531
145王子成,李忠爱,邓秀新.植物反转录转座子及其分子标记[J].植物学通报,2003,20(3):287-294
146 Purugganan M D. Transposon signatures-species-specific molecular markers that utilize a class of multiple-copy nuclear-DNA[J].Mol Ecol,1995,4(2): 265-269
147唐益苗,马有志.植物反转录转座子及其在功能基因组学中的应用[J].植物遗传资源学报, 2005,6(2):221-225
2李志勇,陈凤林,陈立波,等.优良禾草新品种—蒙古冰草[J].中国草地,1993,1:74,78
3李立会,扬新民.通过属间杂交向小麦转移冰草基因的研究[J].中国农业科学, 1998,31(6):1-5
4高卫华,云锦凤,杨静等.冰草属牧草营养器官解剖学研究[J].内蒙古草业,1990,4:19-22
5王怡丹,郭晓宇,全炳武.水分胁迫下蒙古冰草、扁穗冰草和滨麦抗旱性研究[J].延边大学农学学报,2008,30(2):98-104
6王荣华,石雷,汤庚国,等.渗透胁迫对蒙古冰草幼苗保护酶系统的影响[J].植物学通报,2003, 20(3):330-335
7阎贵兴,张素贞.沙芦草染色体组型的分析.中国草地学报,1985,2:38-40
8段晓刚,王丽,卜秀玲.蒙古冰草和布顿大麦染色体组型分析.中国草地,1990(3):45-48
9 Chen Q, Jahidr J,Cauderon Y. Cytological studies on Agropyron Gaertn Species from Inner Mongolia,China[J].C.R.Acad.Sci.SerieⅢ,1989,309(11):519-525
10李立会,徐世雨.冰草属中B染色体在减数分裂期的遗传行为研究[J].植物学报,1991,33(11):833-839
11云锦凤,斯琴高娃.蒙古冰草B染色体的研究[J].内蒙古农牧学院学报,1996,17(1):14-17
12解新明.蒙古冰草(Agropyron mongolicum Keng)遗传多样性研究[D].内蒙古农业大学,2001
13解新明,云锦凤,赵冰,等.蒙古冰草遗传多样性的等位酶分析[J].草业科学,2001,6:6-11
14兰宝祥.蒙古冰草居群遗传多样性研究[J].中国农业科学,2005,38(3):468-473
15云锦凤,李瑞芬.冰草的远缘杂交及杂种分析[J].草地学报,1997,4:221-227
16乌仁其木格,于卓.几种小麦族禾草及其杂种酯酶同工酶研究[J].内蒙古农业大学学报(自然科学版),1999,4:49-53
17王月华,于卓,王四清,等.蒙古冰草×航道冰草杂种F3代的育性及染色体构型分析[J].中国草地,2003,25(4):1-7
18权威,于卓,王月华,等.蒙古冰草×航道冰草杂种F4代株系同工酶分析[J].草地学报,2006,14(1):14-19
19于卓,马艳红,李小雷,等.蒙古冰草与航道冰草正、反交F1及其染色体加倍植株AFLP分析[J].中国草地学报,2009,31(6):14-19
20赵彦,云锦凤.蒙古冰草Lea3抗旱基因片段的分离与鉴定[J].华北农学报,2008,23(6):64-67
21康虹丽.蒙古冰草磷脂酶D基因全长cDNA序列的克隆及生物信息学分析[D].内蒙古农业大学,2008
22何军贤,傅家瑞.种子Lea蛋白的研究进展[J].植物生理学通讯,1996,32(9):291-246
23 Dure L,Greenway SC,Galau GA. Developmental biochemistry of cotton seed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by invitro and invivo protein synthesis[J].Biochemistry,1981,20(14): 4162-4168
24 Grzelczak ZF,Sattolo MH,Hanleybowdoin LK, et al., Synthesis and turnover of proteins and messenger-RNA in germinating wheat embryos[J].Canadian Journal of Biochemistry,1982,60:389-397
25 Bartels D, Singn M, Salamini F. Onset of desiccation tolerance during development of the barley embryo[J]. Planta,1988,175(4): 485-492
26 Bostock RM,Quatrano RS.Regulation of em gene-expression in Rice:Interaction between osmotic-stress and abscisic acid[J].Plant Physiology,1992,98(4): 1356- 1363
27 Campbell SA,Crone DE,Ceccardi TL,et al.A ca.40 kDa maize (Zea mays L.)embryo dehydrin is encoded by the dhn2 locus on chromosome 9[J].Plant Molecula Biology,1998,38(3):417-423
28 Hsing YC,Chen ZY,Shih MD,et al.Unusual sequences of group 3 LEA mRNA inducible by maturation or drying in soybean seeds[J].Plant Mol Biol,1995,29(4):863-868
29 BRAY EA.Molecular responses to water deficit[J].Plant Physiol,1993,103:1035-1040
30 Franz G, Hatzopoulos P,Jones TJ,et al. Molecular and genetic analysis of an embryonic gene, DC 8, from Daucus carota L. Mol Gen Genet, 1989, 218(1): 143-151
31 Prieto DP,Almoguera C,Rojas A, et al. Seed-specific expression patterns and regulation by ABI3 of an unusual late embryogenesis-abundant gene in sunflower[J]. Plant Mol Biol, 1999,39(3):615-627
32 Ingram J,Bartels D.The molecular basis of dehydration tolerance in plants[J].Annu Rev Plant Physiol Plant Mol Biol,1996,47:377-403
33 Hughes DW,Galau GA.Developmental and environmental induction of LEA and LEAA messenger-RNAS and the postabscission program during embryo culture[J].Plant Cell,1991,3(6):605-618
34 Caray Arroyo A,Colmenero Flores JM,Garoiarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit[J].Biological Chemistry,2000,275(8):5668-5674
35 Wise MJ, Tunnacliffe A.POPP the question:what do LEA proteins do?[J].Trends Plant Science,2004,9(1):13-17
36 Hollung K, Espelund M, Jakobsen KS. Another LEA B19 gene (Group1 LEA) from barley containing a single 20 amino acid hydrophilic motif[J]. Plant Molecular Biology, 1994,25(3):559-564
37 Swire Clark GA, Marcotle WR. The wheat LEA protein Em functions as an osmoprotective molecule in Sacharomyces cerevisiae. Plant Biology,1999,39(1):117-128
38 Stacy RAP, Espelund M, Saeboe-Larssen S,et al. Evolution of the Group-1 Late Embryogenesis abundant(LEA) genes analysis of the LEA B19 gene family in barley[J]. Plant Molecular Biology,1995,28(6):1039-1054
39 Bray EA, Alterations in Gene Expression in Response to Water Deficit. In: Basra AS Stress-induced Gene Expression in Plants,Newark:Harwood Academic, 1994, 1-23
40 Koag MC,Wilkens S,Fenton RD,et al. The K-segment of maize DHN1 mediates binding to anionic phospholipid vesicles and concomitant structural changes[J].Plant Physiology,2009,150(3):1503-1514
41宋松泉,陈玲,傅家瑞.种子脱水耐性与LEA蛋白[J].植物生理学通讯,1999,35(5):424-431
42 Rorat T.Plant dehydrins-tissue location, structure and function[J].Cell.Mol. Biol.Lett.,2006,11(4):536-556
43 Danyluk J, Perron A, Houde M,et al. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat[J].Plant cell,1988,10(4):623-638
44 Close TJ. Dehydrins: A commonality in the response of plants to dehydration and low temperature[J].Physiolgia Plantarum.1997,100(2):291-296
45 Curry J, Walter-Simmons MK. Unusual sequence of group 3 LEA(Ⅱ) mRNA inducible by dehydration stress in wheat[J].Plant Molecular Biology,1993,21(5),907-912
46 Straub PF, Shen QX, Ho T-HD. Structure and promoter analysis of an ABA and stress regulated barley gene, HVA1 [J].Plant Mol. Biol., 1994,26(2):617-630
47 Dure L.A repeating 11-mer amino acid motif and plant desiccation[J].Plant Journal,1993,3(3):363-369
48孙海丹,兰英,刘昀,等.LEA蛋白质11-氨基酸基序与植物抗旱性[J].东北师范大学学报:自然科学版,2004,36(3):85-90
49 Kim HS,Lee JH,Kim JJ, et al.Molecular and functional characterization of CaLEA6, the gene for a hydropobic LEA protein from Capsicum annuum[J]. Gene,2005,344: 115-123
50 Maitra N,Cushman JC.Isolation and characterzation of a drought-induced soybean cDNA-encoding a D95 family late-embyogenesis-abundant protein[J].Plant Physiology,1994,106(2):805-806
51 Roberts JK,DeSimone NA.Cellular concentrations and unifomuty of cell-type accumulation of two Lea proteins in cotton embryos.Plant Cell,1993,5: 769-780
52 Goday A,Jensen AB,Culianez-Macia FA,et al.The maize abscisic acid-responsiveprotein Rab 17 is located in the nucleus Cell and interacts with nuclear localization signals[J]. Plant Cell,1994, 6:351-360
53 Asghar R,Fenton RD,DeMason DA,et al.Nuclear and cytoplasmic localization of maize embryo and aleurone dehydrin[J].Protoplasma,1994,171:87-94
54 Close TJ.Dehydrins:Emergence of a biochemical role of a family of plant dehydration protein[J].Physiol Plant,1996,97:795-803
55 Ukaji N,Kuwabara C,Takezawa D,et al.Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3 late-embryogenesis abundant proteins[J].Plant Physiology,2001,12(4)6: 1588-1597
56 Jensen AB, Goday A, Figueras M, et al. Phosphorylation mediates the nuclear targeting of the maize Rab17 protein[J].Plant Journal,1998,13(5):691-697
57 Alsheikh MK,Heyen BJ, Randall SK. Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation [J]. Journal of Biological Chemistry,2003,278(42): 40882-40889
58 Nair R,Rost B. LOCnet and LOCtarget:sub-cellular localization for structural genomics targets[J].Nucleic Acids Research,2004,32:517-521
59 Xu DP,Duan XL,Wang BY,et al.Expression of a late embryogenesis abundant protein gene,HVA1,from barley confers tolerance to water deficit and salt stress in transgenic rice [J].Plant Physiol,1996,110(1):249-257
60 Imai R,Chang L,Ohta A,et al.A lea-class gene of tomato congers salt and freezing tolerance when expressed inSaccharomyces cerevisiae[J].Gene,1996,170:243-248
61俞嘉宁,张林生,张劲松.小麦耐逆基因TaLEA3的克隆及在酵母中的功能分析[J].生物工程学报,2004,26(6),832-838
62赵咏梅,杨建雄,俞嘉宁,等.小麦耐逆基因-TaLEA2转化拟南芥的研究[J].西北植物学报,2006,26(1):1-6
63 Garay-Arroyo A,Colmenero-Flores JM,Garciarrubio A,et al.Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit [J].J Biol Chem,2000,275:5668-5674
64袁克华,冯仁军,程萍,等.RNA干扰的研究进展[J].安徽农业科学,2009,37(8):3471-3473
65 Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811
66刘敏,曹毅,将彦.利用RNAi技术大规模分析基因功能的研究.植物学通报.2002.19(4):491-495
67 Cogoni C,Irelan JT, Schumacher M,et al.Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does notdepend on DNA-DNA interactions or DNA methylation. Embo journal,1996,15(12): 3153-3163
68朱旭东,黄培堂.RNA干涉的研究进展[J].生物技术通讯,2001,12(4):61-65
69石燕.RNAi实验技术研究进展[J].标记免疫分析与临床,2003,10(4):235-238
70 NyKanen A,Haley B,Zamore P D. ATP requirements and small interfering RNA structure in the RNA interference pathway[J].Cell,2001,107(3):309-321
71 Carmell MA.Germline transmission of RNAi in mice[J].Nature Struct.Biol.2003, 10:91-92
72 English JJ,Davenport GF,et al.Requirement of sense transcription for homology dependent virus resistance and trans-inactivation. Plant J,1996,12:597-603
73韩蓓,王秀敏,顾学范.双链RNA干涉技术(RNAi)在不同生物中应用的研究进展[J].遗传,2002,24(2):200-202
74 Wesley SV,Helliwell CA,Smith NA,et al.Construct design for efficient,effective and high-throughput gene silencing in plants.Plant J.200l,27(6):581-590
75朱俊华,竺晓平,温孚江,等.马铃薯Y病毒衣壳蛋白基因片段长度对RNA介导抗病性影响[J].中国科学C辑:生命科学,2004,34(1):23-30
76 Elbashir SM, Martinez J. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J].Embo journal,2001,20(23):6877-6888
77 Schweizer P,Pokorny J,Schulze-Lefert P,et al.Double-stranded RNA interferes with gene function at the single-cell level in cereals[J].Plant Journal,2000,24: 895-903
78 Liu Q,Singh SP,Green AG.High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing[J].Plant Physiology, 2002,129(4):1732-1743
79 Chuang CH,Meyerowitz EM.Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana[J].Proc.Natl.Acad. Sci.USA,2002,97: 4985-4990
80 Chi JT,Chang HY,Wang NN,et al. Genomewide view of gene silencing by small interfering RNAs[J].Proc.Natl.Acad.Sci.USA,2003,100(11):6343-6346
81周小云,陈信波,向建华. RNAi技术及在植物基因功能研究中的应用[J].生物学杂志,2005, 22(2):38-40,9
82徐化学,熊建华,傅彬英.应用Gateway技术构建水稻OsDAD1基因的RNA干涉载体[J].分子植物育种,2007,5(1):133-136
83 GATEWAY-(TM)——一种新型克隆技术[J].微生物学通报,2000,27(5):388
84 Curtis MD , Grossniklaus, U. gateway cloning vector set for high-throughput functional analysis of genes in planta[J].Plant physiology.2003,133(2):462-469
85谈心,杨宏,乔定君,等.干扰烟草GA 20-氧化酶siRNA植物表达载体的构建及矮化烟草的产生[J].应用与环境生物学报,2008,14(1):48-52
86柴晓杰,王丕武,关淑艳,等.应用RNA干扰技术降低玉米支链淀粉含量[J].植物生理与分子生物学学报,2005,31(6):625-630
87陈坚,兰涛,段远霖,等.水稻中SUSIBA2相似基因RNA干扰体系的建立[J].福建农林大学学报(自然科学版),2009,38(1):39-43
88彭昊,翟英,张芊,等.水稻高效RNA干涉体系的建立及其功能分析[J].中国农业科学,2006, 39(9):1729-1735
89刘玉汇.Patatin启动子驱动的马铃薯GBSSⅠ基因ihpRNAi植物表达载体的构建及转化马铃薯的研究[D].甘肃农业大学,2008
90李丽文,朱延明,李杰,等. RNAi技术在植物功能基因组学中的研究进展[J].东北农业大学学报,2007,38(1):119-124
91 Grimsley N, Hohn T, Boulton M L,et al. Agroinfection of Zea mays with maize streak virus DNA[J].Life sciences,1987,140:459-468
92 Ishida Y,Saito H. High efieiency transformation of maize(zea mays L.)mediated by Agrobacterium tumefaeiens[J].Natural Bioteehnol,1996,14(6):745-750
93 Hiei Y,Ohta S,Komari T.Effieient transformation of rice(Oryza sativa L.) mediated by Agrobaeterium and sequence analysis of the boundaries of the T-DNA[J].Plant Journal,1994,6:271-282
94 Sung HP, Shannon RM,Smith RH.T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices[J].Plant Molecular Biology,1996, 32(6):1135-1148
95 Cheng M,Fry JE,Pang SZ,et al. Genetic transformation of wheat mediated by Agro- bacterium tumefaciens[J].Plant Physiol,1997,115:971-980
96夏光敏,李忠谊. Transgeni根癌农杆菌介导的小麦转基因植株再生[J].植物生理学报, 1999,25(1):22-28
97胡张华,吴关庭,金卫.农杆菌介导的水稻转化及bar基因稳定遗传[J].浙江农业学报,2003,15(6):327-331
98肖军,石太渊,郑秀春,等.根癌农杆菌介导的高粱遗传转化体系的建立[J].杂粮作物,2004,24(4):200-203
99佘建明,张保龙,何晓兰,等.草地早熟禾农杆菌介导法基因转化条件[J].草地学报,2005,13(1): 39-43
100李达旭,张杰,赵建,等.根癌农杆菌介导转化川草二号老芒麦胚性愈伤组织[J].植物生理与分子生物学学报,2006,32 (1): 45-51
101胡繁荣.根癌农杆菌介导的金边狗牙根遗传转化条件的优化[J].植物资源与环境学报,2005,14 (2): 15-18
102张振霞,刘萍,杜雪玲,等.农杆菌介导GUS基因对多年生黑麦草转化的研究[J].广西植物,2007,27(1):121-126
103李进,侯海军,黄复深,等.基因枪介导获得转植酸酶基因水稻研究[J].2008,14(1):6-10
104李国圣,杨爱芳,张举仁,等.玉米愈伤组织的遗传转化及抗除草剂植株再生[J].科学通报, 2000,20:2181-2184
105魏松红,张领兵,张艳贞,等.小麦基因枪高效转化体系的建立[J].吉林农业科学,2001,2(3):7-11
106李志亮,黄丛林,王刚,等.利用基因枪法向高羊茅导入bar基因的研究[J].河北师范大学学报(自然科学版),2006,2:217-221
107 Zhou G Y,Weng J,Zeng Y, et al.Introduction of exogenous DNA into cotton embryos[J].Meth. Enzymo.,1983,101:433-481
108黄国存,董越梅,孙敬三,等.以GFP为标记用花粉管通道途径导入外源DNA[J].科学通报, 1998(23): 2531-2533
109武小霞,刘伟婷,刘琦,等.利用花粉管通道法将抗虫基因(cry V)转入大豆的研究[J].2010,29(4):565-568,574
110王秀君,郎志宏,陆伟,等.利用花粉管通道法将耐草甘膦基因mG2-epsps导入玉米自交系的研究初报[J].中国农业科技导报, 2008, 10(4):56-62
111沈世华,张秀君,郭奕明,等.玉米基因转化的离体子房注射及其转基因植株的鉴定[J].植物学报, 2001, 43: 1055-1057
112 Krens FA,Molendijk L,Wullems GJ,et al.In vitro transformation of plant protoplasts with Ti-plasmid[J].Nature,1982,296:72-74
113薛红卫,卫志明,许智宏.通过PEG法转化甘蓝获得转基因植株[J].植物学报,1997,39(1):28-33
114钟玉海,申艮宝.植物基因的遗传转化方法[J].安徽农学通报,2010,16(11):65-66,206
115任永霞,季静.植物遗传转化方法概述[J].河北北方学院学报(自然科学版),2005,21(6):38-42
116 Joersbo M,Brunstedt J.Directgene transfer to plantprotoplasts bymild sonication[J].Plant Cell Reports,1990,(9):207-210
117王关林,方宏筠.植物基因工程[M].科学出版社,2009,7
118余迪求,邓庆丽,沈亚楠,等.金鱼草基因转化和转基因植株再生[J].热带亚热带植物学报,1996,4(4):86-90
119贺竹梅.人促红细胞生成素基因在番茄中的表达[J].遗传学报,1998,25(2):155-159
120许新萍,陈金婷,张建中,等.抗稻瘟病和纹枯病的转基因水稻新品系[J].中山大学学报(自然科学版),2001,40(3):131-132
121 Dewal Jani, Laxman Singh Meena. Expression of cholera toxin B subunit in transgenic tomatoplant[J].Transgenic Research, 2002,11:447-454
122张荃,王淑芳,赵彦修,等.HAL1基因转化番茄及耐盐转基因番茄的鉴定[J].生物工程学报,2001,17(6):658-662
123 Samia Djennane, Jean-Eric Chauvin, Isabelle Quillere, et al. Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers[J].Transgenic Research,2002, 11:175-184
124王莉江,明小天,安成才,等.籼稻明恢63成熟种子愈伤组织的诱导及转基因水稻的抗性检测[J].生物工程学报,2002,18(3):323-327
125吴刚,崔海瑞,舒庆尧,等.转基因水稻中转录水平cry1Ab基因的沉默及其阶段复活[J].中国科学(C辑),2001,31(6):487-496
126曾千春,吴茜,周开达,等.修饰的cry1Ac基因导入籼稻明恢81获得抗虫纯合系[J].遗传学报,2002,29(6):519-524
127 Geng Sa,Ma Mi,Ye He-Chun, et al. Anther-specific expression ofiptgene in transgenic tobacco and its effect on plant development [J]. Transgenic Research,2002, 11: 269-278
128马媛媛.抗旱基因LEA的克隆及其序列解析[D].东北林业大学,2003
128邢浩然,刘丽娟,刘国振.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21:1-6
129 Abelson J, Simon M. Green fluorescent protein[J].Methods in Enzymology,1999,302: 449
130陈建勋,王晓峰.植物生理学实验指导[M].广州:华南理工大学出版社,2002
131高俊凤.植物生理学实验技术[M].世界图书出版社,2000
132 Bates LE,Waldren RP,Teare ID.Rapid determination of free proline for water stress studies[J].Plant Soil,1973,39:205-207
133李合生,孙群,赵世杰,等.植物生理生化实验原理和技术[M].北京:高等教育山出版社, 2000:260
134胡张华,吴关庭,金卫.农杆菌介导的水稻转化及bar基因稳定遗传[J].浙江农业学报,2003,15(6):327-331
135巩健,杨芳.单子叶植物表达载体的构建及农杆菌介导的玉米遗传转化的研究[J].生物技术,2007,17(3):2-5
136梁雪莲,孙毅.农杆菌介导转化小麦幼胚获得抗除草剂再生植株[J].植物生理与分子生物学学报,2003, 29(6): 501-506
137易自力,曹守云,王力,等.提高农杆菌转化水稻频率的研究[J].遗传学报,2001,28(4):352-358
138刘巧泉,张景六,王宗阳.根癌农杆菌介导的水稻高效转化系统的建立[J].植物生理学报,1999,24:259-271
139 Sahi S V,Chilton M D,Chilton W S.Corn metabolites affect growth and virulence of Agrobacterium tumefaciens[J]. Proc Natl Acad Sci USA.1990,27:3879-3883
140 Grandbastien M A. Retroelements in higher-plants[J].Trends Genet.,1992,8:103-108
141 Kunze R, Saedle H, Lonnig WE. Plant transposable elements[J].Adv.Bot.Res.,1997,27: 331-470
142 Voytas DF,Cummings MP,Konerczny AK,et al. Copia-like retrotransposons are ubiquity-ous among plants[J].Proc NAtl Acid Sci USA,1992,89(15):7124-7128
143 Kumar A,Bennetzen J I.Plant retrotransposons[J].Annu Rev Genet,1999,33:479-532
144刘柱,朱建清,赵建,等.植物反转录转座子的研究进展[J].生物化学与生物物理,2002,29 (4):527-531
145王子成,李忠爱,邓秀新.植物反转录转座子及其分子标记[J].植物学通报,2003,20(3):287-294
146 Purugganan M D. Transposon signatures-species-specific molecular markers that utilize a class of multiple-copy nuclear-DNA[J].Mol Ecol,1995,4(2): 265-269
147唐益苗,马有志.植物反转录转座子及其在功能基因组学中的应用[J].植物遗传资源学报, 2005,6(2):221-225