【作者】 韩小花；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 谷类作物种子内淀粉的积累对于作物产量的形成具有极为重要的意义。种子淀粉的生物合成是各种酶催化的过程,这一模型已经初步建立。然而随着越来越多的该模型外的粉质突变体基因的发现,新的多网络交叉模型的建立显得尤为重要。为寻找淀粉合成相关的新基因,我们对实验室已有的日本晴T-DNA插入突变体库和日本晴、93-11辐射诱变突变体库进行了筛选(总共筛选11400份变突变体材料),并鉴定到多份粉质突变体。本文针对其中的一个突变体——T3612,进行详细研究,并取得如下研究进展：T3612种子外观及横截面呈现不透明粉质表型,与野生型日本晴相比较千粒重下降33.7%；突变体种子横截面扫描电镜观察结果显示,T3612淀粉颗粒变圆、变小,而且排列疏松；突变体种子生理生化测定结果显示,蛋白质和脂肪含量上升大约15%,直链淀粉含量是野生型的80%。通过图位克隆的方法,利用T3612/南京11F2群体中1242个粉质表型极端个体,将目的基因定位于第11染色体短臂25cM处,位于OSJNBa0058P12BAC克隆的T3612-54和T3612-31两个标记间的61kb区域。经RICEGAAS预测该区间有11个基因,RT-PCR表明该区间PDIL1-1未表达。基因组序列测序表明PDIL1-1从ATG后124bp处开始总共缺失4145bp。功能互补实验进一步证实了我们所克隆到的基因PDIL1-1就是控制突变性状的关键基因。利用半定量RT-PCR和western blot进行组织表达模式分析,结果表明PDIL1-1呈现组成性表达,在根、茎、叶、鞘、穗和胚乳中均有表达,而且在发育12天的胚乳中表达量最高。但是在突变体T3612中,不论是在RNA水平还是蛋白水平均检测不到PDIL1-1的表达。这进一步确证粉质突变体T3612确实是PDIL1-1缺失型突变体。我们利用荧光定量PT-PCR的方法,检测了31个淀粉合成相关基因的表达情况。与野生型比较,突变体中OsAGPL2、OsPHOL、OsSuSy2和OsSuSy3的表达增加了将近1倍；另外OsAGPL1、OsAGPL4、OsSSIIB,OsPHOH和OsDPE-1的表达降低至野生型的一半。利用非变性淀粉胶电泳后活性染色的方法,我们检测了突变体发育9天和12天胚乳中淀粉合成酶(starch synthase, SS)、淀粉分支酶(branching enzyme, BE)、淀粉磷酸化酶(Starch Phosphorylase, Pho)和淀粉脱分支酶(debranching enzyme, DBE)的活性。活性染色结果显示,普鲁兰酶的活性下降,而且突变体普鲁兰酶条带迁移率下降,ISA的活性没有明显差异；SSⅠ的活性显著上升,而SSⅢ的活性变化不明显；Pho1的活性显著下降,Pho2的活性没有变化；淀粉分支酶的活性均未变化。另外ADPG焦磷酸化酶(AGPase)和蔗糖合酶(Sucrose Synthase)的活性显著增加,UDPG焦磷酸化酶(UGPase)活性变化不大。除此之外,我们利用基于DNA测序的荧光糖电泳(DSA-FACE)的方法测定了突变体种子中支链淀粉链长分布特征。结果8≤DP≤13的链长比例增加,6≤DP≤7的链长比例降低。PDIL1-1编码一个蛋白质二硫键异构酶。体外表达PDIL1-1重组蛋白并进行酶活实验分析,表明我们克隆到的重组PDIL1-1蛋白可以还原胰岛素,而且还原活性与PDIL1-1蛋白的浓度成正比。另外,重组PDIL1-1在体外可以有效地抑制柠檬酸合酶的热变性,说明其具有分子伴侣活性。PDIL1-1通过对底物蛋白二硫键的氧化还原,可以帮助其形成正确构象。荧光定量RT-PCR检测分析发现突变体胚乳细胞中Bip、Hsp70、Hsp90、CRT、CNX等分子伴侣基因,NEF、ERdj3like、Stt3a和UDPG-glucose-transporteor等辅助分子伴侣基因,bZIP转录因子基因bZIP60和ERAD降解途径基因Derlins的表达量均高于野生型。这些基因受内质网胁迫(ER stress)所诱导表达,这说明突变体胚乳细胞内发生内质网胁迫。内质网胁迫可使细胞启动程序性死亡(PCD),本研究中RT-PCR检测PCD相关基因HSR203J在突变体胚乳细胞中的表达明显高于野生型。因此我们推断突变体胚乳细胞内发生严重内质网胁迫从而诱发PCD,进一步影响了胚乳细胞内的淀粉积累。更多还原

【Abstract】 Starch accretion determines final grain weight and thereby it contributes greatly to grain productivity. The enzymatic process model required for starch synthesis has been established by a series of mutants using maize and rice as model organisms. However studies on some new opaque endosperm mutants showed that these new genes can not be added into the previously established model, so we need to construct a bigger or a cross model using these novel mutants. To seek for more these kinds of mutant in rice, we performed a large-scale screen in T-DNA-inserted and60Co-irradiated mutant pool and isolated a series of mutant lines. T3612was one of those. One of the floury mutants T3612was selected for further research and and the detailed research progress which achieved in this study is as follows:T3612showed floury endosperm and the final mature seed weight was～33.7%lower than that of wild type. Scanning electron microscopic (SEM) examination revealed that the mutant endosperms contained round loosely packed starch granules which were probably responsible for the floury appearance. The amylose content in T3612was approximately80%that of Nipponbare. The total protein and lipid content was approximately15%higher than wild type.To genetically map the mutation, an F2population derived from a cross between T3612which is an indica variety, and japonica variety Nanjingll. Furthermore, we used1242floury segregants to located the mutation between marker T3612-31and T3612-54, and the mutation co-segregated with marker T3612-55, locating within a61kb region of BAC clone OSJNBa0058P12on chromosome11. RiceGAAS (http://ricegaas.dna.affrc. go.jp/) predicted eleven ORFs in this map region. The mutation gene was PDIL1-1. DNA-PCR amplification and sequencing revealed a4145bp deletion started from124bp in PDIL1-1CDS. Transgenic complementation test confirmed that the deletion of protein disulfide isomerase like1-1(PDIL1-1) was responsible for the floury phenotype. Expression analysis demonstrated that PDIL1-1was localized in the root, culm, sheath, leaf and greatly had the highest expression level at DAF12. T3612produced no OsPDIL1-1transcript or the OsPDIL1-1protein in those tissues. These results suggested that T3612was a deleted mutant of protein disulfide isomerase like1-1(PDIL1-1).The real-time RT-PCR was used to analyze the expression of31starch synthesis-related genes. Results suggested that genes expression for OsAGPL2、OsPHOL、 OsSuSy2and OsSuSy3, was double that of wild type, while expression of the genes for OsAGPL1、OsAGPL4、OsSSIIb, OsPHOH and OsDPE-1in T3612was decreased to half of those in wild type. Zymogram analyses were performed using T3612and the wild-type Nipponbare endosperm at9DAF and12DAF. We detected the activities of starch synthase (SS), branching enzyme (BE), Starch Phosphorylase (Pho) and debranching enzyme (DBE). The activity of pullulanase decreased largely and migration rate of PUL band decreased, whereas SSI increased significantly. The activity of Pho1was decreased significantly at9DAF. No significant differences in activities of BE isoforms (BEI, BEIIa, and BEIIb), SSIII, Pho1and ISA were found. AGPase and Suc synthase activity was higher than wild type.UGPase activity was also found not to be significantly different in the mutant than in the wild type Nipponbare. Then We determined the chain-length distributions of mutant endosperm amylopectin using DNA sequencer assisted fluorophore carbohydrate electrophoresis (DSA-FACE). The mutant endosperm had a higher proportion of short chains with a degree of polymerization (DP) of8to13and a decrease in chains with DP6to7.PDIL1-1encoded a protein disulfide isomerase. To determine whether PDIL1-1protein show disulfide reductase and molecular chaperone activity, we used insulin and citrate synthetase as substrate in our study. The results suggested that insulin was reduced more rapidly with the increase in the concentration of recombinant PDIL1-1. And recombinant PDIL1-1can effectively protect citrate synthetase from thermal denaturation in42℃in vitro.In the ER lumen, protein disulfide isomerase-like (PDIL) proteins assist in nascent immature secretary proteins folding. We detected the expression levels of genes encoding chaperones and oxidoreductases in mutant endosperm12DAF by employing Real-Time RT-PCR. In our study, ER stress-responsive genes were strongly up-regulated, such as PDIL, Bip, Hsp70, Hsp90, CRT, CNX, cochaperone (NEF, ERdj3, Stt3a and UDPG- glucose-transporteor), bZIP60and Derlins. Persistent or acute ER stress aims to initiate programmed cell death, and the PCD marker gene HSR203J was induced in T3612. So we speculated that programmed cell death which maybe happened in mutant endosperm influenced the starch synthesis.更多还原