【作者】 王颖；

【导师】 刘巧泉； 张昌泉；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2020， 硕士

【摘要】 淀粉是水稻胚乳中最主要的成分,占稻米干重的80%以上。胚乳淀粉的组成和结构是决定稻米品质的重要因素。稻米外观品质是影响稻米商品价值的重要指标,其直接影响稻米在市场上的受欢迎程度。垩白是影响稻米外观品质的重要指标,其遗传调控非常复杂,不仅受内源基因的调控而且还存在复杂的基因与环境互作的调控方式。此外,还涉及水稻源库流的协调。因此,水稻垩白性状一直以来都是稻米品质遗传改良的重点和难点。ADP葡萄糖焦磷酸化酶(AGPase)是参与淀粉合成的关键酶和限速酶,其突变往往对胚乳淀粉合成和稻米品质产生重要影响。在前期研究中,我们通过化学诱变方式获得了一个水稻心白突变体agpl2,通过图位克隆和测序分析发现控制心白突变性状的候选基因为编码水稻AGPase的一个大亚基OsA GPL2基因,agpl2突变体中OsAGPL2基因编码区发生了一个单碱基突变。本研究系统分析了 agpl2突变体稻米的品质特性和造成心白性状的可能机理,主要结果如下:1、序列分析表明突变体中OsAGPL2基因第9外显子上一个碱基由G突变为A,该突变造成了甘氨酸到丝氨酸的变异。农艺性状调查表明,突变体植株较野生型在株高和分蘖数等方面没有显著变化。但突变体稻米表现出极高的垩白粒率和垩白度。此外,突变体的粒长和粒宽稍有增加。通过互补试验,将OsAGPL2过表达构建导入agpl2突变体,发现籽粒胚乳恢复透明,验证了垩白表型是由OsA GPL2基因位点突变所导致的。2、稻米结构分析表明,agpl2突变体为心白突变,胚乳两端透明,心部不透明。扫描电镜观察显示,野生型稻米淀粉颗粒为规则的多面体且淀粉粒之间结合紧密。突变体籽粒透明部分淀粉粒排列规则且紧密,但籽粒中垩白部位淀粉颗粒排列松散,形状不规则,小颗粒较多。通过淀粉粒径分析发现,agpl2淀粉粒的平均粒径显著减小,大淀粉粒的比例明显降低。籽粒灌浆速率分析表明,突变体agpl2较野生型灌浆速率前期较慢但在后期表现出增加的趋势。随着籽粒灌浆的进行,野生型淀粉粒体积增大,形状逐渐规则且排布紧密;突变体agpl2的淀粉粒从灌浆后期开始与野生型出现明显差异,淀粉粒小且形状多呈椭圆形,彼此间空隙较大。3、稻米理化品质分析表明,与野生型对照相比,突变体稻米的蛋白质含量没有显著差异,但是总淀粉含量下降,表观直链淀粉含量上升,胶稠度下降,淀粉粘滞性上升,糊化温度增加,热焓值降低。淀粉精细结构分析表明,突变体agpl2支链淀粉中聚合度(DP)为5-12的短链比例减少,而聚合度(DP)为12-37的支链比例增加,支链淀粉分支度增加。晶体结构分析表明突变体agpl2淀粉的结晶度下降。对花后不同时期FACE分析表明突变体agpl2较野生型分支度一直较高,短支链淀粉含量少而中长链淀粉含量多。4、系统分析了突变体中AGPase各亚基编码基因和其它淀粉合成酶基因的表达特性,结果表明突变体中AGPase各亚基编码基因均有上调,可溶性淀粉合成酶编码基因OsSSI和OsSSIVb表达呈下调趋势;淀粉分支酶编码基因OsBEIIb除花后15天表达上调外,其他时期均低于野生型;灌浆中后期OsSIIa、OsGBSSI、OsBEI、OsISAI、OsPUL表达量上调。Western杂交分析表明OsAGPL2蛋白丰度稍有增加,酶活性定量分析表明突变体agpl2中AGPase酶活性增加。进一步通过酵母双杂分析发现,突变体OsAGPL2蛋白自我结合力减弱而OsAGPL2与OsAGPS2b间的结合力增强。更多还原

【Abstract】 Starch is the most important component in rice endosperm and it occupys over 80%rice dry weight.The composition and structure of endosperm starch are important factors in determining rice grain quality.The rice appearance quality is an important parameter affecting rice commodity value,which directly affects consumer’s preference and choice.The chalkiness is an important parameter of rice appearance,and the genetic regulation of chalky traits is very complex,which is not only regulated by endogenous genes,but also by the interaction of gene and environment.In addition,the source-sink relationships and grain filling can affect the chalkiness.Therefore,chalky traits have always been an important subject for rice grain quality genetic improvement.ADP glucose pyrophosphorylase(AGPase)is a key enzyme and a speed limiting enzyme involved in starch synthesis,and the mutation of AGPase will lead to a serious effect on rice endosperm starch synthesis and thus rice grain quality.In previous studies,we obtained a stable rice white core endosperm mutant agpl2 by chemical mutagenesis.By using Map-based cloning and gene sequencing,we found that the candidate gene controlling the white core mutation is the OsAGPL2 gene which encodes the rice AGPase large subunit.A single base mutation was found in the exon 9 of OsAGPL2 gene in agpl2 mutant.In the present study,the grain quality characteristics of agpl2 mutant was systematically analyzed and the possible mutation mechanism that caused the white core mutation was explored.The main results were as follows:1.Sequence analysis showed that a single base substitution occurred in the exon 9 of agpl2 gene with G to A mutation,and thus lead to the corresponding glycine to serine substitution.The investigation of agronomic characters showed that the agpl2 mutant rice had no significant changes in plant height and tiller number compared with the wild type,while the mutant rice showed extremely high chalky grain rate and chalky degree.In addition,we found that the grain length and width of the mutant increased slightly.2.Grain structure analysis indicated that the agpl2 mutant was a classic white core mutation with transparent endosperm at both ends and opaque core.Scanning electron microscopy(SEM)observation of the rice grain cross section showed that the starch granules from wild type were regular polyhedron and closely bound to each other.Similarly,the starch granules from the transparent part of the mutant grain were arranged regularly and tightly,while starch granules were loosely arranged with irregular shape and more small particles in the central chalky region of the mutant rice.Then the starch granule size distribution was further analyzed.Compared with the wild type,we found that the average particle size of agpl2 starch decreased greatly and also the proportion of large starch granules decreased too.Grain filling rate analysis showed that the mutant agpl2 grains exhibited a slower filling rate than the wild-type in the early stage of seed development,however,an increasing trend in the later stage.SEM observation of the isolated starch from the developing seeds showed that wild-type starch granule size increased and closely arranged as the development of the seed.However,significant differences were found in the late filling stage of the mutant rice.The mutant agpl2 grain showed a smaller irregular starch granule with obvious loosely arrangement in the late filling stage.3.The physicochemical characters of rice grain were analyzed.Compared with the wild type,there was no significant difference in protein content.However,the total starch content,gel consistency(GC)and the enthalpy decreased significantly.While the apparent amylose content(AAC),starch viscosity,and gelatinization temperature(GT)increased significantly.Starch fine structure analysis showed that the amylopectin short chains with polymerization degree(DP)of 5-12 decreased compared with the agpl2 mutant,while the proportion of amylopectin with DP of 12-37 increased.Besides,the branching degree of amylopectin increased.Crystal structure analysis showed that the relative crystallinity of the mutant agpl2 starch decreased.FACE analysis at different periods after flowering showed that the mutant agpl2 had higher branching degree than wild-type,and the content of short amylopectin was less and that of medium long amylopectin was more.4.The expression profiles of AGPase encoding genes and other starch synthesis related genes were analyzed.The results showed that the expression levels of all the AGPase encoding genes increased slightly in the agpl2 mutant.Meanwhile,the soluble starch synthesis genes OsSSI and OsSSIVb showed a decreased expression level.Also,the starch branching enzyme gene OsBEIIb showed a decreased expression trend during the seed developing except 15 days after flowering.Besides,some other starch synthesis related genes such as OsSSIIa,OsGBSSI,OsBEI,OsISA1 and OsPUL all exhibited an increase trend during the late grain filling stage.Western blotting analysis showed that OsAGPL2 protein abundance increased slightly in the agpl2 mutant Further enzyme activity assay showed that the activity of AGPase in agpl2 mutant increased than that in wild type.Then,through yeast two hybrid analysis,we found that the self-binding ability of the mutant OsAGPL2 protein decreased while the binding ability between mutant OsAGPL2 and wild-type OsAGPS2b increased.更多还原