油葵PEPC基因保守序列的克隆及RNAi载体的构建
摘要
油葵(Helianthus annuus L.)是我国五大油料作物之一,具有耐寒耐旱,耐盐碱耐瘠薄,适应性广,对土壤要求不高等特性。早在本世纪50年代,品质遗传学家就有报道称,油菜籽中蛋白质的含量与油脂含量呈高度的负相关关系,在其中起重要作用的关键酶则是磷酸烯醇式丙酮酸羧化酶(PEPC)。现在在油菜和大豆上已经有PEPC敲除实例,其对产油量确实产生了一定的促进作用。
鉴于油葵PEPC基因未知,本研究根据亲缘较近物种的PEPC基因同源克隆了油葵PEPC基因mRNA的部分序列,同源比对找到两个进化保守区,构建RNAi表达载体,以期能够提高油葵的含油量。本研究得到实验结果如下:
(1)用同源克隆的方法得到了油葵PEPC基因以及mRNA的部分序列。由于油葵中对该基因的研究还没有报道,因此需要以此序列再到GENEBANK中进行比对。比对结果显示,所扩增片段与GENEBANK中其他物种PEPC序列同源性极高,尤其是与油葵同科植物黄花菊属和青蒿的同源性为最高,与黄花菊属中不同植物的PEPC基因相似度达到92%以上,说明所得序列可以判断为油葵PEPC基因部分序列。
(2)在油葵PEPC基因中通过比对找到两个保守区,根据RNAi要求dsRNA的大小,以及pTCK-303载体中的酶切位点,设计引物,克隆这两个片段,并连入了T载体测序无误。分别正反向连入载体pTCK-303,成功构建油葵PEPC基因的RNAi载体pTCK303-FP1-RP1和pTCK303-FP2-RP2。
(3)通过比较,确定油葵的组织培养植株再生的最佳外植体为茎尖周缘分生区。其愈伤诱导率和不定芽的诱导率都高于其他外植体。最佳不定芽诱导培养基为MS +IAA(0.03 mg·L~(-1))+6-BA(1.4～1.6 mg·L~(-1)),诱导率可达76.67%。在培养基1/2MS + NAA(0.3 mg·L~(-1))上进行生根培养,所有不定芽均可生根,且须根较多,利于组培苗的移栽成活。经过油葵花粉管通道法转化的探索,确定其可以得到阳性种子,为下一步研究奠定基础。
Oil sunflower is one of the five oilseed crop, with a cold drought, salinity tolerance barren, wide adaptability and poor soil dependent characteristics and so on. Back in the 50s of this century, geneticists have reported that the quality of rapeseed protein and fat content was highly negatively correlated, which plays an important role is phosphoenolpyruvate carboxylase enzyme (PEPC). Now in rape and soybean, PEPC has been knocked and its oil production does have a certain increase.
Since the PEPC gene in oil sunflower hasn’t been reported before, we have cloned its PEPC mRNA partial sequence by alignment with close relatives of this species. Compared with other homologous PEPC genes, two evolutionary conserved regions are found. RNAi expression vectors are constructed with these two regions. We expect to improve oil content in oil sunflower. The results of this study are as follows:
(1) Obtain PEPC gene and partial sequence of mRNA by using homology cloning methods. Comparison to GENEBANK showed that it is highly homologous with the sequences of PEPC in other species, especially with same family (Asteraceae) plants—F.trinervia and Artemisia annua. And the highest homology with F.trinervia PEPC gene is 92% of similarity, and illustrate the sequence can be judged as sunflower PEPC gene sequences.
(2) Find two conserved regions in PEPC gene of sunflower by comparing. According to the size of RNAi requires and pTCK-303 vector restriction sites, design two pairs of primers to clone the two fragments, and link them into the T vector. Then sequence the fragments and ensure it is correct. Positively and negatively, connected it to carrier pTCK-303. Construct RNAi vector pTCK303-FP1-RP1 and pTCK303-FP2-RP2.
(3) By comparison, the best explant in tissue culture is cotyledonary nodes. The callus induction and shoot induction rates are higher than other explants. The best medium for shoot induction is MS + IAA (0.03 mg·L~(-1)) +6- BA (1.4 ~ 1.6 mg·L~(-1)), the induction rate reached 76.67%. In the medium 1/2MS + NAA (0.3 mg·L~(-1)).All adventitious buds can root, and fibrous roots are more conducive to survive the transplanted seedlings. After exploreing transformation of sunflower pollen tube pathway, determine their seeds can be positively transformed, and lay the foundation for further study.
鉴于油葵PEPC基因未知,本研究根据亲缘较近物种的PEPC基因同源克隆了油葵PEPC基因mRNA的部分序列,同源比对找到两个进化保守区,构建RNAi表达载体,以期能够提高油葵的含油量。本研究得到实验结果如下:
(1)用同源克隆的方法得到了油葵PEPC基因以及mRNA的部分序列。由于油葵中对该基因的研究还没有报道,因此需要以此序列再到GENEBANK中进行比对。比对结果显示,所扩增片段与GENEBANK中其他物种PEPC序列同源性极高,尤其是与油葵同科植物黄花菊属和青蒿的同源性为最高,与黄花菊属中不同植物的PEPC基因相似度达到92%以上,说明所得序列可以判断为油葵PEPC基因部分序列。
(2)在油葵PEPC基因中通过比对找到两个保守区,根据RNAi要求dsRNA的大小,以及pTCK-303载体中的酶切位点,设计引物,克隆这两个片段,并连入了T载体测序无误。分别正反向连入载体pTCK-303,成功构建油葵PEPC基因的RNAi载体pTCK303-FP1-RP1和pTCK303-FP2-RP2。
(3)通过比较,确定油葵的组织培养植株再生的最佳外植体为茎尖周缘分生区。其愈伤诱导率和不定芽的诱导率都高于其他外植体。最佳不定芽诱导培养基为MS +IAA(0.03 mg·L~(-1))+6-BA(1.4～1.6 mg·L~(-1)),诱导率可达76.67%。在培养基1/2MS + NAA(0.3 mg·L~(-1))上进行生根培养,所有不定芽均可生根,且须根较多,利于组培苗的移栽成活。经过油葵花粉管通道法转化的探索,确定其可以得到阳性种子,为下一步研究奠定基础。
Oil sunflower is one of the five oilseed crop, with a cold drought, salinity tolerance barren, wide adaptability and poor soil dependent characteristics and so on. Back in the 50s of this century, geneticists have reported that the quality of rapeseed protein and fat content was highly negatively correlated, which plays an important role is phosphoenolpyruvate carboxylase enzyme (PEPC). Now in rape and soybean, PEPC has been knocked and its oil production does have a certain increase.
Since the PEPC gene in oil sunflower hasn’t been reported before, we have cloned its PEPC mRNA partial sequence by alignment with close relatives of this species. Compared with other homologous PEPC genes, two evolutionary conserved regions are found. RNAi expression vectors are constructed with these two regions. We expect to improve oil content in oil sunflower. The results of this study are as follows:
(1) Obtain PEPC gene and partial sequence of mRNA by using homology cloning methods. Comparison to GENEBANK showed that it is highly homologous with the sequences of PEPC in other species, especially with same family (Asteraceae) plants—F.trinervia and Artemisia annua. And the highest homology with F.trinervia PEPC gene is 92% of similarity, and illustrate the sequence can be judged as sunflower PEPC gene sequences.
(2) Find two conserved regions in PEPC gene of sunflower by comparing. According to the size of RNAi requires and pTCK-303 vector restriction sites, design two pairs of primers to clone the two fragments, and link them into the T vector. Then sequence the fragments and ensure it is correct. Positively and negatively, connected it to carrier pTCK-303. Construct RNAi vector pTCK303-FP1-RP1 and pTCK303-FP2-RP2.
(3) By comparison, the best explant in tissue culture is cotyledonary nodes. The callus induction and shoot induction rates are higher than other explants. The best medium for shoot induction is MS + IAA (0.03 mg·L~(-1)) +6- BA (1.4 ~ 1.6 mg·L~(-1)), the induction rate reached 76.67%. In the medium 1/2MS + NAA (0.3 mg·L~(-1)).All adventitious buds can root, and fibrous roots are more conducive to survive the transplanted seedlings. After exploreing transformation of sunflower pollen tube pathway, determine their seeds can be positively transformed, and lay the foundation for further study.
引文
[1] D. H. PUTNAM,E. S. OPLINGER,D. R. HICKS,et al. Sunflower. Alternative filed crops manual,1990: 53-54
[2]中国农业百科全书·农作物卷·下.农业出版社,1995: 592
[3]刘学彬,王霞明,殷松枝.杂交油葵高产栽培技术.农牧产品开发,1996(7): 39-40
[4] J. V. ABELARDO,H.D. IAN,SCOTTC C. Progress over 20 years of sunflower breeding in central Argentina. Field Crops Research,2007,100(1): 61-72
[5] RONDAN,A. MANTESE,R. SAVIN,et al. Responses of sunflower yield and grain quality to alternating day/night high temperature regimes during grain filling: effects of timing,duration and intensity of exposure to stress. Field Crops Research,2008,96(1): 48-62
[6]安玉麟.中国向日葵产业发展的问题与对策.内蒙古农业科技,2004,(4): 1-4
[7]王燕飞,于伯成,向理军等.油用型向日葵籽实含油率与气候因素的关系.中国油料作物学报,1997,4(3): 45-49
[8]刘兆普,隆小华,刘玲,等.海岸带滨海盐土资源发展能源植物资源的研究.自然资源学报,2008,23(1): 11-12
[9] E. BELHASSEN,G. AUGE. Dynam icmanagement of genetic resources: first generation analysis of sunflower artificial populations. Genet Sel Evol,1994,26(1): 241-253
[10]刘公社,彭克敬,王树年.杂交油葵高产栽培技术问答.北京:科学普及出版社,1997: 10-96
[11]邓力群,刘兆普,程爱武,等.不同盐分滨海盐土上油葵(G1012B)的氮磷肥效应研究.中国油料学报,2002,24(4): 35-41
[12]崔云玲,王生录,陈炳东.油葵耐盐性试验研究.甘肃农业科技,2002(8): 35-38
[13]李卫华,郝乃斌,戈巧英等. C3植物中C4途径的研究进展.植物学通报,1999,16(2): 97–106
[14] R. CHOLLET,J. VIDAL,M.H. LEARY. Phosphoenolpyruvate carboxylase: a ubiquitous,highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular Biology,1996,47: 273-298
[15]陈锦清,郎春秀,胡张华,等.反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究.农业生物技术学报,1999,7(4): 316-320
[16]潘瑞炽,董愚得.植物生理学.第三版.北京:高等教育出版社,1995: 67-115
[17]陈锦清,黄锐之,郎春秀,等.油菜PEP基因的克隆及PEP反义基因的构建.浙江大学学报(农业与生命科学版),1999,25(4): 365-367
[18]张银波,江木兰,胡小加.油菜PEPase基因的克隆及其对应RNAi载体的构建.中国油料作物学报,2005,27(1): 1-4
[19]汪承刚,谢好,李长春,等.甘蓝型油菜三种种子储藏蛋白和丙酮酸羧化酶基因片段的克隆及hpRNA表达载体的构建.中国油料作物学报,2006,28(3): 245-250
[20] A. FIRE,S. XU,M. K. MONTGOLNERY,et al. Potent and specific genetic interfere by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391: 806-811
[21] P. A .SHARP,P. D. ZARFLORE. RNA interference. Science,2000,287: 2431-2433
[22] L. HEATHER,CHOTKOWSKI,T. ALEXANDER,et al.West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.Virology,2008,377: 197-206
[23] M. BUHLER,D. MOAZED. Transcription and RNAi in heterochromaticgene silencing . Nature Mol Biol,2007,14(11): 1041-1048
[24] D. KRISTIN,KASSCHAU,NOAH FAHLGREN,et al. Genome-Wide Profiling and Analysis of Arabidopsis siRNAs. PLoS Biology,2007,5(3): 479-493
[25] Y. TOMARI,P. D. ZAMORE. Perspective: Machines for RNAi. Genes,2005,19: 517-529
[26] Z. KLASE,P. KALE,R.WINOGRAD,et al. H IV-1 TAR element is processed by dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BM C Mol Biol,2007,8: 63
[27] G. MEISTER,T. TUSCHL. Mechanisms of gene silencing by double-stranded RNA. Nature,2004,431: 343-349
[28] M. A. Behlke. Progress towards in vivo use of siRNAs. Mol Ther,2006,13 (4) : 644 - 70
[29] E. BERNSTEIN,C. D. ALLIS. RNA meets chromatin. Genes,2005,19: 1635-1655
[30] Y. SENECHAL,P. KELLY,F. JOHN,et al . siRNA-mediated knock-down of the serotonin transporter in the adult mouse brain. Neurochemistry,2007,102 (6): 1928
[31] P. BRODERSEN,O.VOINNET.The diversity of RNA silencing pathways in plants. Trends Genet,2006,22: 268-280
[32] H. VAUCHERET. Post-transcriptional small RNA pathways in plants: Mechanisms and regulations. Genes,2006,20: 759-771
[33] O. PONTES,C. F. LI,P. C. NUNES,et al. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell,2006,126: 79-92
[34] T. KANNO,B. HUETTEL,M. F. METTE,et al. Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet,2005,37: 761-765
[35] HUNG CHUAN-FU,LU KUANG-CHU,CHENG TSUNG-LIN,et al. A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells. Biochemical and Biophysical Research Communications,2006,346: 707-720
[36]汤华. RNA干扰原理与应用.北京:科学出版社,2006: 9
[37]乔枫,樊颖伦,杨清,等. RNAi分子机制及在植物功能基因组研究中的应用.生物技术通报,2006(4): 20-24
[38] S. TRAVELLA,T. E. KLIMM,B. KELLER. RNA Interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat. Physiology,2006,142: 6-20
[39]赵明敏. RNA干扰及其在植物研究中的应用.中国农学通报,2006,22(4): 36-39
[40] A. M. CIGAN,E. UNGER WALLACE,HAUG COLLET,et al. Source transcriptional gene silencing as a tool for uncovering gene function in maize. Plant J,2005,43: 929-940
[41] L. L. LI,L. Q. RAO,SH. FAN,et al. Progress of the technology of RNAi andits aplication in plant RNAi. Journal of Hunan Environment Biological Poly technic,2005,11(4): 313-318
[42] J. M. VARQASON,G. SZITTYA,Y. J. BURQ,et al. Size selective recognition of siRNA by an RNA silencing suppressor. Cell,2003,115(7): 799-811
[43] P. L. GA,F. L. CAREY,C. N. PEDRO,et al. The arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell,2006,126 (1): 79-92
[44] C. TRAN,P. D. ZAMORE. Small silencing RNAs. Curr. Biol.,2007,17(18): 789-793
[45] Q. LIU,S. .P SINGH,A. G. Green. High-stearieand High-oil cotton seed Produced by hairpin RNA mediated Post-transeriPtional gene sileneing. Plant,2002,129: 1732-1743
[46] S. OGITA,H. UEFUJI,Y. Yube,et al. Producing decaffeinated coffee Plants. Nature,2003,423: 428
[47] J. R. LI,W. ZHAO,Q. Z. LI.,et al. RNA silencing of waxy gene results in low levels of amylose in the seeds of transgenic wheat. Journal of Genetics and Genomics,2005,32(8): 846-854
[48] G. R.DAVULURI,A. V. TUINEN,P. D. FRASER,et al. Fruit specific RNAi mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat. Biotechnol,2005,23(7): 825-826
[49] A. S. XIONG,Q. H. YAO,R. H. PENG,et al. Different effect on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato . Plant Cell,2005,23: 639-646
[50] E. FUKUSAKI,K. KAWASAKI,S. KAJIYAMA,et al. Flower color modulations of Torenia hybrida by down regulation of chalcone sythase genes with RNA interference. J Biotechnol, 2004,111(3): 229-240
[51]杨小二. RNA技术及其在植物分子生物中的应用.分子植物育种,2005,3(4): 571-574
[52]赵晓波,葛嶙,林红.高产优良油用向日葵新品种-S31.新疆农业科学,2002,(2): 17
[53]闵恩泽.以植物油为原料发展我国生物柴油炼油厂的探讨.石油学报(石油加工),2005,21(3): 25-28
[54]梅德清,袁银南,孙平,等.植物油作为发动机燃料及生物柴油制备的研究.小型内燃机与摩托车,2005,34(3): 24-27
[55]刘宫社,阿兰·博让,彭克敬,等.向日葵研究与开发.北京:中国科学技术出版社,1994:54-132
[56] Y. KAI,H. MATSUMURA,K. IZUI. Phosphonenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. Arch Biochem Biophy,2003,414: 170-179
[57] K. IZUI. Phosphoenolpyruvate carboxylase: a new era of structuralbiology. Annu Rev Plant Biol,2004,55: 67-84
[58] P. A. STOUTJESDIJK,S .P. SINGH,C. J. HURLSTONE,et al. hpRNA-mediated targeting of the Arabidiosis FAD2 gene gives highly efficient and stable silencing. Plant Physiol,2002,129(4): 1723-1731.
[59]陈彦.花粉管通道导入外源DNA方法的研究.北方园艺,2010,13: 226-228
[60]李向龙,张力全,张晓东.花粉管通道法在玉米转基因育种中的应用.种子科技,2010,03: 23-24
[61]王映红,刘小片,洪得峰.花粉管通道法在转基因中应用.农技服务,2009,26(6): 9-15
[62]马盾,周小云,黄乐平.花粉管通道法转基因棉花后代的遗传特性.新疆农业科学,2008 ,17(3): 147-149
[63]韦佳,张寅寅,陈鹏彦.硝酸银对蕨菜和朝鲜野菊花愈伤组织生长与分化的影响.科技信息,2009,11: 42-43
[2]中国农业百科全书·农作物卷·下.农业出版社,1995: 592
[3]刘学彬,王霞明,殷松枝.杂交油葵高产栽培技术.农牧产品开发,1996(7): 39-40
[4] J. V. ABELARDO,H.D. IAN,SCOTTC C. Progress over 20 years of sunflower breeding in central Argentina. Field Crops Research,2007,100(1): 61-72
[5] RONDAN,A. MANTESE,R. SAVIN,et al. Responses of sunflower yield and grain quality to alternating day/night high temperature regimes during grain filling: effects of timing,duration and intensity of exposure to stress. Field Crops Research,2008,96(1): 48-62
[6]安玉麟.中国向日葵产业发展的问题与对策.内蒙古农业科技,2004,(4): 1-4
[7]王燕飞,于伯成,向理军等.油用型向日葵籽实含油率与气候因素的关系.中国油料作物学报,1997,4(3): 45-49
[8]刘兆普,隆小华,刘玲,等.海岸带滨海盐土资源发展能源植物资源的研究.自然资源学报,2008,23(1): 11-12
[9] E. BELHASSEN,G. AUGE. Dynam icmanagement of genetic resources: first generation analysis of sunflower artificial populations. Genet Sel Evol,1994,26(1): 241-253
[10]刘公社,彭克敬,王树年.杂交油葵高产栽培技术问答.北京:科学普及出版社,1997: 10-96
[11]邓力群,刘兆普,程爱武,等.不同盐分滨海盐土上油葵(G1012B)的氮磷肥效应研究.中国油料学报,2002,24(4): 35-41
[12]崔云玲,王生录,陈炳东.油葵耐盐性试验研究.甘肃农业科技,2002(8): 35-38
[13]李卫华,郝乃斌,戈巧英等. C3植物中C4途径的研究进展.植物学通报,1999,16(2): 97–106
[14] R. CHOLLET,J. VIDAL,M.H. LEARY. Phosphoenolpyruvate carboxylase: a ubiquitous,highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular Biology,1996,47: 273-298
[15]陈锦清,郎春秀,胡张华,等.反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究.农业生物技术学报,1999,7(4): 316-320
[16]潘瑞炽,董愚得.植物生理学.第三版.北京:高等教育出版社,1995: 67-115
[17]陈锦清,黄锐之,郎春秀,等.油菜PEP基因的克隆及PEP反义基因的构建.浙江大学学报(农业与生命科学版),1999,25(4): 365-367
[18]张银波,江木兰,胡小加.油菜PEPase基因的克隆及其对应RNAi载体的构建.中国油料作物学报,2005,27(1): 1-4
[19]汪承刚,谢好,李长春,等.甘蓝型油菜三种种子储藏蛋白和丙酮酸羧化酶基因片段的克隆及hpRNA表达载体的构建.中国油料作物学报,2006,28(3): 245-250
[20] A. FIRE,S. XU,M. K. MONTGOLNERY,et al. Potent and specific genetic interfere by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391: 806-811
[21] P. A .SHARP,P. D. ZARFLORE. RNA interference. Science,2000,287: 2431-2433
[22] L. HEATHER,CHOTKOWSKI,T. ALEXANDER,et al.West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.Virology,2008,377: 197-206
[23] M. BUHLER,D. MOAZED. Transcription and RNAi in heterochromaticgene silencing . Nature Mol Biol,2007,14(11): 1041-1048
[24] D. KRISTIN,KASSCHAU,NOAH FAHLGREN,et al. Genome-Wide Profiling and Analysis of Arabidopsis siRNAs. PLoS Biology,2007,5(3): 479-493
[25] Y. TOMARI,P. D. ZAMORE. Perspective: Machines for RNAi. Genes,2005,19: 517-529
[26] Z. KLASE,P. KALE,R.WINOGRAD,et al. H IV-1 TAR element is processed by dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BM C Mol Biol,2007,8: 63
[27] G. MEISTER,T. TUSCHL. Mechanisms of gene silencing by double-stranded RNA. Nature,2004,431: 343-349
[28] M. A. Behlke. Progress towards in vivo use of siRNAs. Mol Ther,2006,13 (4) : 644 - 70
[29] E. BERNSTEIN,C. D. ALLIS. RNA meets chromatin. Genes,2005,19: 1635-1655
[30] Y. SENECHAL,P. KELLY,F. JOHN,et al . siRNA-mediated knock-down of the serotonin transporter in the adult mouse brain. Neurochemistry,2007,102 (6): 1928
[31] P. BRODERSEN,O.VOINNET.The diversity of RNA silencing pathways in plants. Trends Genet,2006,22: 268-280
[32] H. VAUCHERET. Post-transcriptional small RNA pathways in plants: Mechanisms and regulations. Genes,2006,20: 759-771
[33] O. PONTES,C. F. LI,P. C. NUNES,et al. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell,2006,126: 79-92
[34] T. KANNO,B. HUETTEL,M. F. METTE,et al. Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet,2005,37: 761-765
[35] HUNG CHUAN-FU,LU KUANG-CHU,CHENG TSUNG-LIN,et al. A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells. Biochemical and Biophysical Research Communications,2006,346: 707-720
[36]汤华. RNA干扰原理与应用.北京:科学出版社,2006: 9
[37]乔枫,樊颖伦,杨清,等. RNAi分子机制及在植物功能基因组研究中的应用.生物技术通报,2006(4): 20-24
[38] S. TRAVELLA,T. E. KLIMM,B. KELLER. RNA Interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat. Physiology,2006,142: 6-20
[39]赵明敏. RNA干扰及其在植物研究中的应用.中国农学通报,2006,22(4): 36-39
[40] A. M. CIGAN,E. UNGER WALLACE,HAUG COLLET,et al. Source transcriptional gene silencing as a tool for uncovering gene function in maize. Plant J,2005,43: 929-940
[41] L. L. LI,L. Q. RAO,SH. FAN,et al. Progress of the technology of RNAi andits aplication in plant RNAi. Journal of Hunan Environment Biological Poly technic,2005,11(4): 313-318
[42] J. M. VARQASON,G. SZITTYA,Y. J. BURQ,et al. Size selective recognition of siRNA by an RNA silencing suppressor. Cell,2003,115(7): 799-811
[43] P. L. GA,F. L. CAREY,C. N. PEDRO,et al. The arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell,2006,126 (1): 79-92
[44] C. TRAN,P. D. ZAMORE. Small silencing RNAs. Curr. Biol.,2007,17(18): 789-793
[45] Q. LIU,S. .P SINGH,A. G. Green. High-stearieand High-oil cotton seed Produced by hairpin RNA mediated Post-transeriPtional gene sileneing. Plant,2002,129: 1732-1743
[46] S. OGITA,H. UEFUJI,Y. Yube,et al. Producing decaffeinated coffee Plants. Nature,2003,423: 428
[47] J. R. LI,W. ZHAO,Q. Z. LI.,et al. RNA silencing of waxy gene results in low levels of amylose in the seeds of transgenic wheat. Journal of Genetics and Genomics,2005,32(8): 846-854
[48] G. R.DAVULURI,A. V. TUINEN,P. D. FRASER,et al. Fruit specific RNAi mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat. Biotechnol,2005,23(7): 825-826
[49] A. S. XIONG,Q. H. YAO,R. H. PENG,et al. Different effect on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato . Plant Cell,2005,23: 639-646
[50] E. FUKUSAKI,K. KAWASAKI,S. KAJIYAMA,et al. Flower color modulations of Torenia hybrida by down regulation of chalcone sythase genes with RNA interference. J Biotechnol, 2004,111(3): 229-240
[51]杨小二. RNA技术及其在植物分子生物中的应用.分子植物育种,2005,3(4): 571-574
[52]赵晓波,葛嶙,林红.高产优良油用向日葵新品种-S31.新疆农业科学,2002,(2): 17
[53]闵恩泽.以植物油为原料发展我国生物柴油炼油厂的探讨.石油学报(石油加工),2005,21(3): 25-28
[54]梅德清,袁银南,孙平,等.植物油作为发动机燃料及生物柴油制备的研究.小型内燃机与摩托车,2005,34(3): 24-27
[55]刘宫社,阿兰·博让,彭克敬,等.向日葵研究与开发.北京:中国科学技术出版社,1994:54-132
[56] Y. KAI,H. MATSUMURA,K. IZUI. Phosphonenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. Arch Biochem Biophy,2003,414: 170-179
[57] K. IZUI. Phosphoenolpyruvate carboxylase: a new era of structuralbiology. Annu Rev Plant Biol,2004,55: 67-84
[58] P. A. STOUTJESDIJK,S .P. SINGH,C. J. HURLSTONE,et al. hpRNA-mediated targeting of the Arabidiosis FAD2 gene gives highly efficient and stable silencing. Plant Physiol,2002,129(4): 1723-1731.
[59]陈彦.花粉管通道导入外源DNA方法的研究.北方园艺,2010,13: 226-228
[60]李向龙,张力全,张晓东.花粉管通道法在玉米转基因育种中的应用.种子科技,2010,03: 23-24
[61]王映红,刘小片,洪得峰.花粉管通道法在转基因中应用.农技服务,2009,26(6): 9-15
[62]马盾,周小云,黄乐平.花粉管通道法转基因棉花后代的遗传特性.新疆农业科学,2008 ,17(3): 147-149
[63]韦佳,张寅寅,陈鹏彦.硝酸银对蕨菜和朝鲜野菊花愈伤组织生长与分化的影响.科技信息,2009,11: 42-43