1、玉米粉質胚乳突變體的研究宋任濤上海大學玉米是重要的作物和優(yōu)秀的研究模式 Walbot and Evans (2003) Nature Reviews Genetics, 4: 369-379玉米籽粒是“胚乳型”種子的代表玉米籽粒關鍵發(fā)育時期Sabelli and Larkins, Plant Physiol 149 (2009) 14-26 淀粉粒(S)和蛋白體(P)是主要的儲藏形式籽粒是玉米主要的儲藏器官SSP胚乳是主要的儲藏組織蛋白體（PB）由內質網(wǎng)（ER）形成Herman and Larkins (1999) Plant Cell, 11: 601-613醇溶蛋白有序地包裝在蛋白體中Ad

2、apted from Lending and Larkins (1989) Plant Cell 玉米粉質胚乳突變體是早年重要的遺傳突變體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 Univ Agric Exp Stn

3、Memoir 180:1-83, described 40 classic genetic loci.粉質胚乳突變體與高賴氨酸玉米 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 pattern of maize en

4、dosperm 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).玉米已知的粉質胚乳突變體Recessive opaque mutants (o1,o2, o5, o7, o9o11, o13o16),Semi-dominant floury

5、 mutants (fl1, fl2 and fl3)Dominant mutants (Mc and De-B30) Bryan C. Gibbon and Brian A. Larkins (2005) Trends Genet 21, 227-233. 已經克隆的玉米粉質胚乳突變體MutantProductFunctionReferenceo2bZIP transcription factorDown-regulation 22KDa zein, LKR, and etc.Schmidt, et al. (1987) Sciencefl222KDa-zeinWith a defectiv

6、e SP, accumulation on ERColeman, et al. (1995) PNASDe-B3019KDa-zeinWith a S15P mutation in the SP, un-cleavedKim, et al. (2004) Plant Physiol.Mc16-kDa -zeinA frame-shift mutation (38-bp deletion), changed the last 63 amino acidsKim, et al. (2006) Plant J.fl1A novel ER proteinA-to-G point mutation,re

7、sulting in a Ser-to-Glysubstitution in the first transmembrane domainHolding, et al. (2007) Plant Cellopaque7 和opaque6opaque7 (o7) 突變體著名的高賴氨酸突變體McWhirter, KS (1971) A floury endosperm, high lysine locus on chromosome 10. MNL 45:184 Misra, PS et al. (1972) Endosperm protein synthesis in maize mutants

8、 with increased lysine content. Science 176:1425-1426Burr, 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 genomic organization. J Cell Biol 94:201-206 (o2, fl2, o7) 突變體種子蛋白普遍下降zeins

9、33.4%, non-zeins15.1% , total proteins20.7%透射電鏡（TEM）分析o7wt2500 x6000 x21 DAP蛋白體的統(tǒng)計分析蛋白體變少變小對O7基因的圖位克隆 Gene2 appeared to be the candidate gene for o7. 28kb 轉基因功能互作驗證 The cloned gene is the O7 gene. 轉基因RNAi分析Suppression of O7 gene function by RNAi reproduced the opaque endosperm phenotype. Acyl-CoA Sy

10、nthetase (ACS) superfamily Acyl Activating Enzyme (AAE) family O7 編碼酰基活化酶3（AAE3）基因Substrate screening for purified GST-O7. O7 是 Oxalyl-CoA Synthetase 草酸ABO7 蛋白分布在細胞質中CFP-PTS1GFP-O7O7在籽粒中高度表達 No significant difference at transcript level between mutant and wild type. 突變造成O7蛋白顯著減少The 12-bp deletion in

11、 o7 mutant allele caused significant reduction of o7 protein in developing seeds. O7突變影響氨基酸生物合成主要路徑up-regulateddown-regulatedunchangedO7突變在水稻中同樣有效CK NTG NTG NTG TG TG TG opauqe6(proline1)突變體籽粒主要儲藏成分均顯著下降The content of total protein, zein, starch and fat of wt and mutantWT 21DAPSTPBmu 21DAPSTPBo6 mut

12、ant endosperm has greatly reduced storage organelles (protein bodies and starch granules) 淀粉粒和蛋白體顯著變小O6基因的圖位克隆 7個等位突變體確認基因O6編碼 P5CS21-Pyrroline-5-carboxylate synthetaseSzabados and Savour. (2009) Trands in Plant science負責胞質中的脯氨酸合成xO6主要分布在細胞質中The subcellular localization of O6 proteinOnion CellsTobac

13、co CellsO6具有P5CS 酶活 The enzyme activity analysis of recombinant O6 protein O6突變影響脯氨酸生物合成 The free and total proline content of wt and mutant kernel脯氨酸缺乏不影響儲藏蛋白轉錄水平 The expression analysis of zeins in wt and mutant endosperm但儲藏蛋白翻譯水平嚴重下降The expression analysis of zeins in wt and mutant endosperm15KDa

14、16KDa50KDa27KDa22KDa19KDaTublin18DAP21DAP24DAP27DAPWTpro1WTpro1WTpro1WTpro1胚乳蛋白整體翻譯水平下降The total protein analysis of wt and pro1 mutant endosperm of different allele翻譯關鍵調控因子eIF2磷酸化調控 TublineIF2-PeIF2-Total27DAP24DAPWTmuWTmu突變體eIF2磷酸化水平急劇上升，造成翻譯抑制O6突變表型的形成機制P5CSProlineXUncharged tRNAproTranslational

15、stresseIF2phosphorylationProtein synthesisZein基因Zein蛋白蛋 白 體結構基因 fl2, Mc1, DeB30轉錄層面 o2（轉錄因子）翻譯層面 o7, o6（氨基酸供給） o1（蛋白體形成） fl1（蛋白體裝配）粉質胚乳突變體與儲藏蛋白調控儲運層面致 謝Maize Genetics Stock CenterBrookhaven National Laboratory Dr. Frances BurrChina Agricultural UniversityDr. Jinsheng LaiInstitute of Genetics and Developmental Biology, CASDr. Lihuang ZhuUniversity of ArizonaDr. Brian LarkinsS