玉米粉质胚乳突变体.ppt

1、玉米粉质胚乳突变体的研究,宋任涛 上海大学,玉米是重要的作物和优秀的研究模式,Walbot and Evans (2003) Nature Reviews Genetics, 4: 369-379,玉米籽粒是“胚乳型”种子的代表,玉米籽粒关键发育时期,Sabelli and Larkins, Plant Physiol 149 (2009) 14-26,淀粉粒(S)和蛋白体(P) 是主要的储藏形式,籽粒是玉米主要的储藏器官,S,S,P,胚乳是主要的储藏组织,蛋白体（PB）由内质网（ER）形成,Herman and Larkins (1999) Plant Cell, 11: 601-613,醇

2、溶蛋白有序地包装在蛋白体中,Adapted from Lending and Larkins (1989) Plant Cell,玉米粉质胚乳突变体,是早年重要的遗传突变体,First report by Singleton WR and Jones DF in the early of last century, including opaque endosperm1 and opaque endosperm2. Emerson RA, Beadle GW and Fraser AC(1935) A summary of linkage studies in maize. Cornell U

3、niv Agric Exp Stn Memoir 180:1-83, described 40 classic genetic loci.,粉质胚乳突变体与高赖氨酸玉米,Mertz, ET et al. (1964) Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science 145:279-280 (o2). Nelson, OE Jr et al. (1965) Second mutant gene affecting the amino acid

4、 pattern of maize endosperm proteins. Science 150:1469-1470 (floury2 (fl2), o1 and o2). Misra, PS et al. (1972) Endosperm protein synthesis in maize mutants with increased lysine content. Science 176:1425-1426 (bt2, fl2, o2 and o7).,玉米已知的粉质胚乳突变体,Recessive opaque mutants (o1,o2, o5, o7, o9o11, o13o16

5、), Semi-dominant floury mutants (fl1, fl2 and fl3) Dominant mutants (Mc and De-B30),Bryan C. Gibbon and Brian A. Larkins (2005) Trends Genet 21, 227-233.,已经克隆的玉米粉质胚乳突变体,opaque7 和opaque6,opaque7 (o7) 突变体,著名的高赖氨酸突变体,McWhirter, KS (1971) A floury endosperm, high lysine locus on chromosome 10. MNL 45:18

6、4 Misra, PS et al. (1972) Endosperm protein synthesis in maize mutants with increased lysine content. Science 176:1425-1426 Burr, FA and Burr, B (1982) Three mutations in Zea mays affecting zein accumulation: a comparison of zein polypeptides, in vitro synthesis and processing, mRNA levels, and geno

7、mic organization. J Cell Biol 94:201-206 (o2, fl2, o7),突变体种子蛋白普遍下降,zeins33.4%, non-zeins15.1% , total proteins20.7%,透射电镜（TEM）分析,o7,wt,2500 x,6000 x,21 DAP,蛋白体的统计分析,蛋白体变少变小,对O7基因的图位克隆,Gene2 appeared to be the candidate gene for o7.,28kb,转基因功能互作验证,The cloned gene is the O7 gene.,转基因RNAi分析,Suppression

8、of O7 gene function by RNAi reproduced the opaque endosperm phenotype.,Acyl-CoA Synthetase (ACS) superfamily Acyl Activating Enzyme (AAE) family,O7 编码酰基活化酶3（AAE3）基因,Substrate screening for purified GST-O7.,O7 是 Oxalyl-CoA Synthetase,草酸,A,B,O7 蛋白分布在细胞质中,CFP-PTS1,GFP-O7,O7在籽粒中高度表达,No significant diffe

9、rence at transcript level between mutant and wild type.,突变造成O7蛋白显著减少,The 12-bp deletion in o7 mutant allele caused significant reduction of o7 protein in developing seeds.,O7突变影响氨基酸生物合成主要路径,up-regulated,down-regulated,unchanged,O7突变在水稻中同样有效,CK NTG NTG NTG TG TG TG,opauqe6(proline1)突变体,籽粒主要储藏成分均显著下降,

10、The content of total protein, zein, starch and fat of wt and mutant,o6 mutant endosperm has greatly reduced storage organelles (protein bodies and starch granules),淀粉粒和蛋白体显著变小,O6基因的图位克隆,7个等位突变体确认基因,O6编码 P5CS2 1-Pyrroline-5-carboxylate synthetase,Szabados and Savour. (2009) Trands in Plant science,负责

11、胞质中的脯氨酸合成,x,O6主要分布在细胞质中,The subcellular localization of O6 protein,Onion Cells,Tobacco Cells,O6具有P5CS 酶活,The enzyme activity analysis of recombinant O6 protein,O6突变影响脯氨酸生物合成,The free and total proline content of wt and mutant kernel,脯氨酸缺乏不影响储藏蛋白转录水平,The expression analysis of zeins in wt and mutant

12、endosperm,但储藏蛋白翻译水平严重下降,The expression analysis of zeins in wt and mutant endosperm,胚乳蛋白整体翻译水平下降,The total protein analysis of wt and pro1 mutant endosperm of different allele,翻译关键调控因子eIF2磷酸化调控,突变体eIF2磷酸化水平急剧上升，造成翻译抑制,O6突变表型的形成机制,P5CS,Proline,X,Uncharged tRNApro,Translational stress,eIF2phosphorylat

13、ion,Protein synthesis,Zein基因,Zein蛋白,蛋 白 体,结构基因 fl2, Mc1, DeB30,转录层面 o2（转录因子）,翻译层面 o7, o6（氨基酸供给）,o1（蛋白体形成）,fl1（蛋白体装配）,粉质胚乳突变体与储藏蛋白调控,储运层面,致 谢,Maize Genetics Stock Center Brookhaven National Laboratory Dr. Frances Burr China Agricultural University Dr. Jinsheng Lai Institute of Genetics and Developmental Biology, CAS Dr. Lihuang Zhu University of Arizona Dr. Brian Larkins,Shanghai University Dr. Gang Wang Dr. Guife