摘要
水稻是重要的粮食作物,在水稻矮化育种的进程中,仅有少数几个矮源得到了利用,这些矮源所携带的矮秆基因主要是半矮秆基因sd1或其等位基因,同一矮秆基因的利用潜伏着由遗传单一而带来的风险。因此,筛选和鉴定新的具有育种利用价值的半矮秆基因已成为水稻育种实践中非常重要的研究课题。
本研究中的水稻矮秆材料矮泰引-3系由半矮秆籼稻品种泰引1号自然突变而来,我们从矮泰引-3中分离、鉴定到一个新的半矮秆基因sdt2,遗传分析表明,矮泰引-3的矮生性受2对独立遗传的隐性半矮秆基因控制,分别为sd1和sdt2。
为了克隆sdt2基因,我们利用矮泰引-3与南京6号及中花11的F_2、F_3及F_4群体进行了精细定位。该基因首先被定位于水稻第4染色体RM1305和RM5633之间,遗传距离分别是3.8 cM和0.4 cM。进一步扩大群体,对sdt2基因进行精细定位研究。利用已经公布的水稻基因组序列,在sdt2基因附近区域寻找微卫星序列并发展新的标记,利用其中10个有多态性的标记,对群体进行标记检测,最终将sdt2基因精细定位于SSR标记Chr4-48和SSR404之间,遗传距离分别为0.009cM和0.04cM。在这两个标记之间,SSR398、SSR406和Chr4-60与sdt2基因表现为共分离。精细定位的结果表明,sdt2基因紧邻第4染色体的着丝点。根据已有的信息,将sdt2基因的精细定位遗传图与第4染色体的物理图谱进行了整合。
根据sdt2基因定位区域的基因注释信息,采用候选基因策略,共确定了11个预测基因作为sdt2基因的候选基因。根据前面已有的研究结果,我们重点对1个糖基转移酶基因GTase1进行了分析。DNA测序结果表明,GTase1基因在距起始密码子ATG上游830bp处插入了25bp的序列,导致了该基因在矮泰引-3中的表达水平要明显比南京6号中弱。通过PLACE分析,推测可能是由于短序列的插入而导致了顺式作用元件之间的互作发生了变化,从而使得糖基转移酶基因GTase1在突变体中的表达受到了影响。
为了进一步验证GTase1是sdt2基因的候选基因的可能性,我们进行了功能互补实验。将GTase1基因即基因Ⅰ和另一个糖基转移酶基因即基因Ⅱ的全长基因序列导入到A-3/N6和A-3/Z11的回交后代中,其中转GTase1基因的植株的株高明显增加了。综合上述结果,我们可以推断糖基转移酶基因GTase1是sdt2基因的候选基因。而在接下来的过表达实验中,转AK059031和AK100189的中花11与对照相比,株高也明显增加,根变得粗壮。但是我们同时也发现转AK059031的转基因植株的结实率明显降低,而转AK100189的转基因植株的穗粒数要明显增多。同时,我们又利用Clustalx软件构建了粳稻中糖基转移酶第1家族所有基因成员的系统发生树,结果显示有cDNA序列对应的4个糖基转移酶基因:基因Ⅰ即GTase1基因、基因Ⅲ和Ⅳ属于同一亚类,亲缘关系比较近,而基因Ⅱ属于另一亚类,和它们的亲缘关系相对比较远。
Rice (Oryza sativa L.) is the most important staple foods in the world. During the process of dwarf breeding, several kinds of dwarf gene sources have been used. Moreover, genetic analysis had revealed that the dwarf genes of these sources were allelic to sd-1. The frequent use of these single semi-dwarf genes might cause genetic vulnerability to pests and diseases. Therefore, identifying and developing new useful semi-dwarf genes is becoming a crucial subject for practical rice breeding.
In this study, we described the identification and characterization of a new rice semi-dwarf gene obtained from Aitaiyin-3, a natural dwarf mutant from rice cultivar Taiyin-1.Genetic analysis showed that its dwarfism was controlled by two recessive semi-dwarf genes, sd1 and a new semi-dwarf gene sdt2.
In order to clone sdt2, two F2 populations of A-3//N_6 and A-3//zhonghua 11 plus their F3, F4 offsprings were totally established for fine mapping the sdt2 gene .It was found to be roughly located between two SSR markers RM1305 and RM5633 on chromosome 4 with the genetic distances of 3.8 cM and 0.4 cM, respectively. Then we enlarged the populations and according to the known rice genomic sequences, developed new SSR markers. Then using 10 polymorphic markers, the sdt2 was further mapped between two SSR markers Chr4-48 and SSR404, with genetic distances of 0.009 cM and 0.04 cM, respectively. In addition, SSR398, SSR406 and Chr4-60 were found to co-segregate with sdt2 locus. The mapping result showed that sdt2 gene was tightly adjacent to the centromere of chromosome 4. Based on this finding, the high-resolution genetic map of sdt2 gene was integrated with the physical map on chromosome 4.
In accordance with the annotation data in the sdt2 gene-encompassing region, a candidate-gene strategy was adopted to surmise 11 genes as the candidate genes of sdt2. Based on earlier research in our laboratory, one glycosyltransferase gene namely GTase1 was identified. DNA sequenceing analysis indicated that this gene had a insertion of 25 bp at the upstream of the start codon ATG with 830 bp length, leading to the gene expression level obviously lower in mutant A-3 than that in N6. Based on the PLACE results, a short sequence insertion was presumed to change the interactions among cis-acting regulatory elements, which effected the expression of this gene.
Complementary test was employed to further identify GTase1 as the candidate gene of sdt2. Firstly ,full sequences of glycosyltransferase genes involved GTase1 (geneⅠ) and another one (geneⅡ) were introduced to the back-cross offerings of A-3/N6 and A-3/Z11. Subsequently, the height of transformed plants of geneⅠsignificantly increased. According to the analysis above, we could conclude that the GTase1 was the candidate gene of sdt2. As a result of follow up overexpression test, the height of zhonghua11 plants transformed with full cDNA length AK059031 and AK10089 evidently increased, and the roots became thicker comparing to the control. Simultaneously, we also discovered the seeding rate of transgenic plants with AK059031 obviously decreased, but the spikelet numbers of plants transformed AK100189 increased significantly. Furthermore, the phylogenetic tree was constructed of glycosyltransferase family 1 gene members in japonica rice by Clustalx. The results suggested that the genetic relationships among geneⅠ,ⅢandⅣmentioned in our research were closer, and they were attributed to one sub-family,while the geneⅡwhich was far from the three might belong to another family.
引文
[1] Parnell FR, Rangswani GN, Ayyanggar CRS. The inheritance of characters in rice[J] Agri India Bot Ser,1922,11:185-208.
[2] 明峰正夫. 稻に与ける矮性の遗传任就て.日本学术协会报告,1925.
[3] Chang TT. Semi-dwarfing genes in rice germplasm collection[J]. Rice Genetics Newsletter, 1984,1:94-95.
[4] Takahashi ME and Takeda K. Type and grouping of internode pattern in rice culm. (Genetical studies on rice plant, XXXVⅢ) [J]. Memoris Fac Agr Hokkaido Univ,1969,7:32-44.
[5] Takeda K. and Takahashi ME. Preliminary report on cross experiment in six types of internode pattern in rice culm. (Genetical studies on rice plant, XXXVⅢ) [J]. Memoris Fac Agr Hokkaido Univ,1969,7:44-50.
[6] Takeda K. Studies on the character expression and inheritance of dm-type internodedistribution pattern in dwarf rice plants[J].Bull Fac Agr Hokkaido Univ,1974,22:19-30.
[7] Takeda K. Internode elongation and dwarfism in some gramineous plants[J]. Gamma Field Symp, 1977,16:1-18.
[8] Sasaki A, Ashikari M, Ueguchi-Tanaka M, et al. Green revolution: a mutant gibberellin- synthesis gene in rice[J].Nature,2002,416(6882):701-2.
[9] Monna L, Kitazawa N, Yoshino R, et al. Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis[J]. DNA Res,2002,9(1):11-7.
[10] Itoh H, Ueguchi-Tanaka M, Sentoku N, et al. Cloning and functional analysis of gibberellin 3β-hydroxylase genes that are differently expressed during the growth of rice[J].Proc Natl Acad Sci USA,2001,98:8909-8914.
[11] Ashikari M, Wu J, Yano M, Sasaki T, et al. Rice gibberellin-insensitive dwarf mutant gene Dwarf1 encodes the alpha-subunit of GTP-binding protein[J].Proc Natl Acad Sci USA,1999, 96:10284-10289.
[12] Sasaki A, Itoh H, Gomi K, Ueguchi-Tanaka M, et al. Accumulation of the phosphorylatedrepressor for GA signaling in an F-box mutant[J].Science,2003,299:1896-1898.
[13] Ikeda A, Ueguchi-Tanaka M, Sonoda Y, et al. slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8 [J]. Plant Cell ,2001,13:999-1010.
[14] Ueguchi-Tanaka M, Ashikari M, Itoh H, et al. Characterization of rice dwarf mutant, GIBBERELLIN-INSENSITIVE DWARF1 (GID1). In Proceedings of the 17th International Conference on Plant Growth Substances[J]. Brno, Czech Republic,2001.
[15] Müssig C, Fischer S and Altmann T. Brassinosteroid-regulated gene expression[J]. PlantPhysiol,2002,129:1241-1251.
[16] Hong Z, Ueguchi-Tanaka M, Umemura K, et al. A rice brassinosteroid -deficient mutant, ebisu dwarf(d2), is caused by a loss of function of a new member of cytochrome P450[J]. Plant Cell,2003,15(12):2900-10.
[17] Tanabe S, Ashikari M, Fujioka S, et al. A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length[J].Plant Cell,2005,17(3):776-90.
[18] Hong Z, Ueguchi-Tanaka M, Shimizu-Sato S, et al. Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem[J].Plant J,2002,32(4):495-508.
[19] Mori M, Nomura T, Ooka H, et al. Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis[J].Plant Physiol,2002,130(3):1152-61.
[20] Yamamuro C, Ihara Y, Wu X, et al. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint[J]. Plant Cell,2000, 12(9): 1591-606.
[21] Sato Y, Sentoku N, Miura Y, et al. Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants[J]. The EMBO J,1999, 18(4):992-1002.
[22] Ishikawa S, Maekawa M, Arite T, et al. Suppression of tiller bud activity in tillering dwarfmutants of rice[J].Plant Cell Physiol,2005,46(1):79-86.
[23] Hedden P, Phillips AL. Gibbereltin metabolism: New insights revealed by the genes [J]. Trends Plant Sci, 2000 ,5: 523-530.
[24] Yamaguchi, S. Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.)[J].Plant J,1996,10:203-213.
[25] Helliwell CA, Sheldon CC, Olive MR, et al. Cloning of the Arabidopsis ent-kaurene oxidase gene GA3[J]. Proc Natl Acad Sci USA,1998:9019-9024.
[27] Itoh H, Tatsumi T, Sakamoto T, et al. A rice semi-dwarf gene, Tan-Ginbozu(D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase[J]. Plant Mol Biol, 2004,54(4):533-47.
[26] Davidson SE, Smith JJ, Helliwell CA, et al. The pea gene LH encodes ent-kaurene oxidase[J]. Plant Physiol,2004,134(3):1123-34.
[28] Lange T. Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli[J]. Proc Natl Acad Sci USA,1997,94: 6553-6558.
[29] Xu YL, Li L, Wu K, et al. The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression[J]. Proc Natl Acad Sci USA,1995,92(14):6640-4.
[30] Sasaki A, Ashikari M, Ueguchi-Tanaka M, et al. Green revolution: a mutant gibberellin-synthesis gene in rice[J].Nature,2002,416(6882):701-2.
[31] Yamaguchi S, Smith MW, Brown RG, et al. Phytochrome regulation and differential ex-pression of gibberellin 3β-hydroxylase genes in germinating Arabidopsis seeds[J]. Plant Cell,1998,10(12):2115-2126.
[32] Gomi K and Matsuoka M. Gibberellin signaling pathway[J]. Curr Opin Plant Biol,2003,6(5): 489-493.
[33] Jacobsen SE and Olszewski NE. Mutations at the SPINDLY locus of Arabidopsis alter gibberellin signal transduction[J].Plant Cell,1993,5:887-896.
[34] Peng J, Richards DE, Hartley NM, et al. ‘Green Revolution’ genes encode mutant gibberellin response modulators[J]. Nature,1999,400:256-261.
[35] Ikeda A, Ueguchi-Tanaka M, Sonoda Y, et al. slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8 [J].Plant Cell ,2001,13:999-1010.
[36] Fridborg I, Kuusk S, Robertson M, et al. The Arabidopsis protein SHI represses gibberellin responses in Arabidopsis and barley[J].Plant Physiol,2001,127(3):937-48.
[37] van der Knaap E, Kim JH, Kende H. A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth[J]. Plant Physiol,2000,122(3):695-704.
[38] Ueguchi-Tanaka M, Fujisawa Y, Kobayashi M, et al. Rice dwarf mutant d1, which is defective in the a subunit of the heterotrimeric G protein, affects gibberellin signal transduction[J].Proc Natl Acad Sci USA,2000,97:11638-11643.
[39] Ogas J, Kaufmann S, Henderson J, et al. PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis[J]. Proc Natl Acad Sci USA,1999,96:13839-13844.
[40] Sasaki A, Ashikari M, Ueguchi-Tanaka M, et al. Screening of rice GIBBERELLIN-INSENSITIVE DWARF 1 mutants (GID1). In Proceedings of the 17th International Conference on Plant Growth Substances[J].Brno,Czech Republic,2001.
[41] 王克夷. 糖基转移酶研究进展.生物化学与生物物理学进展, 1994, 21: 9-13.
[42] Joe Ross, Yi Li, Eng-Kiat Lim, et al. Higher plant glycosyltransferases. Genome Biology 2001,2(2):reviews 3004.1-3004.6.
[43] Ralston E J, English J J,Dooner H K. Sequence of three bronze alleles of maize and correlation with the genetic fine structure. Genetics,1988,119:185-197.
[44] Szerszen J B, K Szczyglowski, Bandurski S. A gene from Zea mays involved in conjugation of growth hormone indol-3-acetic acid. Science, 1994, 265: 1699-1701.
[45] Lee, H.I, Raskin I.Purification, cloning,and expression of a pathogen inducible UDP-glucose: salicylic acid glucosyltransferase from tobacco. J Biol Chem, 1999, 274: 36637-36642.
[46] Kapitonov D, Yu R K. Conserved domains of glycosyltransferases. Glycobiology, 1999, 9:961～978.
[47] Li Y, Baldauf S, Lim EK, et, al. Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. J Biol Chem, 2001, 276: 4338～4343.
[48] Shibata H., Y. Sawa, T.Oka, S. Sonoke, K. K. Kim, and M. Yoshioka. 1995. Steviol and steviol-glycoside: glucosyltransferase activities in stevia rebaudiana Bertoni-purificatio and partial characterization. Archives of Biochemistry and Biophysics 321:390-396.
[49] Hedden P,Phillips AL. Gibberellin metabolism: New insights revealed by the genes. Trends Plant Sci, 2000,5:523-530.
[50] Huang S, Raman AS, Ream JE Fujiwara H, Cerny RE, Brown SM. Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis, Plant Physiol, 1998,118: 773-781.
[51] Schomberg FM. Bizzell CM. Lee DJ. Zeevaart JAD. Amasino RM. overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell,2003,15:151-163.
[52] Sakamoto T, Kamiya N, Ueguchi-Tanaka M, Iwahori S, Matsuoka M. KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem. Gene Dev, 2003, 15:58-590.
[53] Curtis, I.S., D.A.Ward, S.G.Thomas, A.L.Phillips, M.R.Davey, J.B. Induction of dwarfism intransgenic Solanum dulcamara by overexpression of a gibberellin 20-oxidase cDNA from pump kin.The Plant Journal, 2000,23: 329-338.
[54] Coles JP, Phillips AL, Crokers SJ, Garcia-Lepe R,Lewis MJ, Hedden P. Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J, 1999, 17:547-556.
[55] Hayashi H, Alia Mustardy L, Deshnium P, Ida M, and Murata N. Transformation f Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J. 1997,12:133-142.
[56] J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯. 分子克隆.北京:科学出版社,1992: 954-960.
[57] 郑 霏琴 , 王 宗 阳 , 高 继 平 , 等 . 水 稻 胚 乳 总 RNA 的 分 离 [J]. 植 物 生 理 学 通讯,1993,29(6):438-440.
[58] 赵祥强. 水稻半矮秆基因sdt2的鉴定、精细定位与候选基因筛选.2005年博士论文:113-117.
[59] Kim GT and Tsukaya H. Regulation of the biosynthesis of plant hormones by cytochrome P450s [J].J Plant Res,2002,115(3):169-77.
[60] Chia T,Thorneycroft D,Chapple A,et al.Acytosolic glucosytransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night[J].Plant J,2004,37(6):853-63.
[61] Bijayalaxmi M,S.P.T.Krishnan,S, Vladimir B. Detection and Preliminary Analysis of Motifs in Promoters of Anaerobically Induced Genes of Different Plant Species[J].Annals of Botany 2005.
[62] Continho PM, Deleury E, Davies GJ,et al.An evolving hierarchical family classification for glycosyltransferases[J].J.Mol.Biol,2003,328:307-317.
[63]Chia T, Thorneycroft D, Chapple A, et al. A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night[J].Plant J,2004,37(6):853-63.
[64] Richman AS, Gijzen M, Starratt AN, et al.Diterpene synthsis in Stevia rebaudiana: recruitment and up regulation of key enzymes from the gibberellin biosynthetic pathway[J].Plant J,1999,19:411-421.
[65] Putu S, Tsutomu S, Hidenari S, Masahiro N, Masayuki N, et al. Development of Simple and Efficient in Planta Transformation Method for Rice(Oryza sativa L.) Using Agrobacterium tumefaciens[J]. Journal of Bioscience and Bioengineering, 2005,100 (4):391-397.
[66] 刘巧泉,王宗阳,顾铭洪,等. 根癌农杆菌介导水稻高效转化系统的建立.植物生理学报,1998,24:259-271.
[67] Hiei Y, Ohta S, Toshithro K, et al. Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant Journal,1994,6:271-282.
