影响稻米理化品质微效基因的定位与效应解析

发布时间：2018-01-24 00:08

本文关键词： 水稻染色体片段代换系稻米品质微效QTL定位淀粉结构 SSI基因　出处：《扬州大学》2016年博士论文　论文类型：学位论文

【摘要】：水稻是我国主要的粮食作物之一。稻米品质是稻米作为商品流通与消费过程中的一种综合评价,反映了稻米本身的物理及化学特性。稻米品质属于典型的质量-数量性状,在主效基因控制的基础上还存在一些微效基因。利用高覆盖度的籼粳间染色体片段代换系(CSSL)鉴定微效QTL,优势突出。随着粳稻品种日本晴和籼稻品种9311基因组测序工作的完成,以及高密度分子标记图谱的构建,CSSL群体的构建变得易行,为深入挖掘影响稻米品质的微效QTL或基因提供了便利,从而能为稻米品质改良提供新的基因资源。本研究利用以籼稻品种9311为供体、粳稻品种日本晴为受体构建的整套CSSL为材料,考察了群体稻米品质主要理化指标,借助重测序鉴定的精确基因型图谱,利用ICiMappingv2.2软件定位到一批相关的QTL,同时发现5个QTL具有一因多效,对定位到含这些QTL的3个关键家系进一步分析,通过淀粉分子结构的剖析,探索QTL效应对淀粉结构的影响。为稻米品质的形成及其变异提供丰富的遗传机理。以此同时,利用转基因材料和近等基因系等遗传材料对参与胚乳淀粉合成的跚基因等位变异的效应解析。主要研究结果如下：1、利用染色体片段代换系定位稻米理化品质性状的微效QTL粳稻品种日本晴和籼稻品种9311都含有相同的服b等位基因,因此利用以这两个亲本为受体和供体的染色体片段代换系进行品质分析,可避免因该主效基因差异对稻米品质的影响。本研究重点以已构建的127个染色体片段代换系为材料,通过全基因组重测序分析鉴定其基因型,利用ICiMapping v2.2软件定位到了一批与稻米品质相关的微效QTL。1)稻米理化品质是一个综合性状,各性状间存在复杂的关系。由于不同的研究者所用的实验材料和分析方法各不相同,并且性状本身在遗传表达上较复杂,因此对稻米的遗传控制有着不同的解释。本研究对影响稻米理化品质的7个品质性状14个参数进行了QTL分析,在两个环境下共检测到35个微效QTL,其中19个微效QTL受环境影响较小,能稳定存在。部分QTL与前人报道的结果相同。还有一批QTL如qGC 7.1和qGT12.1与实验室前期研究结果一致,QTL定位区间得到进一步缩小。2)检测到5个具有多效性的QTL,即一个QTL位点(簇)对多个稻米品质性状有效应。其中3个QTL簇与淀粉的理化性质有关。第一个QTL簇位于第2染色体上,与直链淀粉、糊化温度和冷胶粘度的遗传有关,即qAC2.1、qGT2.1、qCPV2.1位于相同的位点,此位点在代换系N132中检测到；第二个QTL簇在第3染色体上,控制着峰值粘度,胶稠度、糙米蛋白含量的遗传,即qPKV3.1、qGC3.1和qBRPC3.1位于同一位点,此位点在代换系N29中检测到；第三个QTL簇位于第6染色体上,控制着糙米和精米蛋白质含量的遗传,即qBRPC6.1与qMRPC6.1位于相同的位点,在多个代换系中(N24、N39、N66、 N67)检测到。第四个QTL簇位于第7染色体上,与稻米蛋白质含量遗传有关,即qBRPC7.1与qMRPC7.1位于相同的位点,在多个代换系中(N48、N60、N62、N73、N88)检测到。第五个QTL簇在第12染色体上,与直链淀粉、糊化温度、峰值粘度和冷胶粘度的遗传相关,即控制直链淀粉的叫C12.1、控制糊化温度的qGT12.1、控制峰值粘度的qPKV12.1以及控制冷胶粘度的qCPV12.1位于相同的染色体片段上,在代换系N127中检测到。2、利用3个重点染色体片段代换系分析重要QTL形成的淀粉结构基础与受体亲本相比,含有多个定位QTL的3个关键染色体片段代换系(N29、N127和N132)成熟种子淀粉的糊化起始温度降低,吸热峰前移,此结果与RVA谱的结果一致。淀粉的DSC相应特征值数据显示,它们对应的峰值温度、终止温度、热焓值,较受体亲本均有所降低,接近于供体亲本9311。淀粉精细结构研究发现,含有来自9311代换片段的N29、N127和N132,其稻米淀粉含有较少的A链和短B链,推测短分支的支链淀粉A链和B1链含量的减少可能由QTL效应引起。综合分析,3个极端家系稻米淀粉可能由于含有较少的A链和短B链、较多的中长链组分,共同抑制了淀粉粒的膨胀,从而导致了稻米理化性质的变化。3、水稻SSI基因等位变异的遗传效应分析可溶性淀粉合成酶基因SSI在水稻理化品质形成中的作用并不大,属微效基因。但其确切的效应并没有被详细解析。本研究利用含不同SSI等位基因(籼稻、粳稻)来源的SSI-RNAi材料及近等基因系,对不同SSI等位基因的表达、对稻米理化品质与淀粉结构的效应等进行了祥细分析。结果显示,SSI在籼粳间的两个主要等位基因(SSI j和SSIi)表达的差异引起了稻米淀粉结构的微妙变化,进而表现为对稻米理化品质有不同的效应。对含不同SSI等位基因的水稻品种分别抑制SSI基因后,稻米的理化品质产生了显著的影响。SSI-RNAi转基因材料直链淀粉含量极显著提高,峰值粘度,崩解值呈极显著降低。不同SSI等位基因遗传背景下,SSI-RNAi材料的表现也存在一定的差异。含SSI j等位基因的粳稻背景下,RNAi系稻米的消碱值比未转化亲本高,达到了极显著差异；而在SSI i等位基因的籼稻背景下,RNAi系稻米的消碱值比亲本要低许多。RVA谱的峰值时间在籼稻RNAi系与亲本间达到了极显著差异,而在粳稻背景下没有差异。这也进一步暗示了不同SSI等位基因对稻米品质遗传的效应是不同的。对稻米淀粉的DSC分析表明,SSI-RNAi材料与其未转化亲本相比,起始温度都显著下降,表现为提前糊化,热晗值都下降。淀粉结晶度数据表明,RNAi系相对亲本而言,淀粉结晶度下降。说明干扰SSI基因表达后能引起淀粉晶体的改变。推测SSI基因在RNAi系中表达量降低后,可能引起淀粉结晶度下降,最终影响稻米的理化品质及食味值。由GPC分析结果推测SSIj等位基因可能负责淀粉中长链的合成,一旦受干扰,淀粉结构中中链长组份减少,SSIi则有可能负责低分子量链的合成。在转基因系中可以看到当SSIi被抑制后,低分子量淀粉分子组分减少。利用近等基因系,我们发现在相同的日本晴(NIP)遗传背景下,不同SSI等位基因(SSIj和SSIi)表达存在差异。轮回亲本(NIP-SSIj)和其近等基因系(NIP-SSIi稻米的直链淀粉含量相近,近等基因系NIP-NIL-SSIi稻米RVA谱比轮回亲本高,发现峰值粘度极显著增加,热浆粘度显著下降,崩解值显著提高。这也暗示着籼、粳稻稻米品质之间的差异,除受Wx主效基因影响外,还与SSI等位基因的变异有一定的联系。DSC分析表明,SSI等位基因的变异导致了起始温度显著下降,表现为提前糊化,而峰值粘度、终止时间、热晗值与轮回亲本相比,均出现一定程度的下降。SSI等位基因的变异未能引起淀粉的结晶度的变化。上述结果表明,当龙特甫中的SSIi导入日本晴后,近等基因系中SSI等位基因表达发生了改变,随之引起低分子量短链的增加。这说明SSI等位基因在籼粳稻淀粉链长的分布中功能存在差异,同时意味着SSI等位基因间的变异导致籼粳稻之间支链淀粉结构产生差异。综合上述研究及取得的结果,一是通过以粳稻为遗传背景的染色体片段代换系在两年两个环境下的实验,对影响稻米理化品质的14个参数进行了QTL分析,共定位了35个QTL,其中5个位点具有一因多效；对含多个定位QTL的3个重要家系的淀粉结构进行了剖析,为解析CSSL稻米品质的差异提供了很好的参考；二是利用RNAi转基因系和近等基因系进一步深入解析了栽培稻中SSI基因两个主要等位变异对稻米品质和淀粉结构的效应。
[Abstract]:Rice is one of the main food crops in China. The quality of rice is rice as a comprehensive evaluation of commodity circulation and consumption process, reflects the physical and chemical properties of rice itself. The rice quality belong to quantitative traits typical quality, based on the major gene control there are some minor genes with high. The coverage between Indica and japonica chromosome segment substitution lines (CSSL) identification of minor QTL advantages. With the japonica cultivar Nipponbare and indica cultivar 9311 genome sequencing was completed, and the construction of high-density molecular marker linkage map construction, CSSL group becomes easy, provides convenience for mining deeply affect the rice quality of micro effect QTL or gene, which can provide new gene resources for improving rice quality. This study using 9311 indica rice varieties as donors, Nipponbare CSSL receptor for the whole construction material for test To observe the main physical and chemical indexes of rice quality groups, accurate genotype map by re sequencing, to a number of related QTL by ICiMappingv2.2 software, also found that 5 QTL has a pleiotropic, 3 key lines containing these QTL positioning for further analysis, through the analysis of the molecular structure of starch and explore the effect of QTL on the structure of starch. Provide rich genetic mechanism for the formation of rice quality and its variation. At the same time, the use of transgenic materials and near isogenic lines and other genetic materials to participate in endosperm starch synthesis to allelic variation effect analysis. The main results are as follows: 1, minor QTL japonica cultivar Nipponbare and indica cultivar 9311 using chromosome segment substitution lines in rice quality traits of physicochemical positioning all contain the same B allele, so using the two parent receptor and donor chromosomes. Segment substitution line quality analysis, can avoid the influence of different genes on rice quality. 127 chromosome segment substitution lines in this study focuses on the building materials, through the analysis of whole genome sequencing to identify the genotype, the use of ICiMapping V2.2 software to locate a number of related micro effect and the quality of rice QTL.1) physico-chemical quality is a comprehensive character, there is a complex relationship between the characters. As the experimental materials and analysis methods used by different researchers have different traits, and genetic expression in itself is complex, so the genetic control of rice have a different interpretation. In this study, physical and chemical quality the effect of rice 7 quality traits of the 14 parameters of the QTL analysis, in the two environments were detected in 35 minor QTL, one of the 19 minor QTL is less affected by the environment, can exist stably. Part QTL and former newspaper The same results. There are a number of QTL such as qGC 7.1 and qGT12.1 and the previous research results, further narrowing the interval QTL positioning.2) detected 5 pleiotropic QTL, namely a QTL locus (clusters) have multiple effects on rice quality traits. The physicochemical properties of 3 QTL clusters and the starch. The first QTL cluster located on chromosome second, and amylose, genetic, gelatinization temperature and cool viscosity is qAC2.1, qGT2.1, qCPV2.1 in the same site, the site in the substitution lines detected in N132; second QTL cluster on chromosome third, controls the peak viscosity, gel consistency, genetic, protein content of brown rice, namely qPKV3.1, qGC3.1 and qBRPC3.1 located in the same site, the site in the substitution lines detected in N29; third QTL cluster located on chromosome sixth, the genetic control of brown rice and rice protein content, namely qBRPC6.1 and qMRPC6.1 On the same site, in a number of substitution lines (N24, N39, N66, N67) were detected. The fourth QTL cluster located on chromosome seventh, and protein content in rice genetic, namely qBRPC7.1 and qMRPC7.1 located on the same site, in a number of substitution lines (N48, N60, N62, N73, N88) detected. Fifth QTL clusters on chromosome twelfth, and amylose content, gelatinization temperature, peak viscosity and cool viscosity of genetic correlation, namely the control of amylose gelatinization temperature control named C12.1, qGT12.1, qPKV12.1 and qCPV12.1 peak viscosity control control of cold glue viscosity located on the same chromosome fragment, to detect.2 in substitution lines N127, using 3 key chromosome segment substitution lines analysis of the formation of QTL starch structure foundation and the recipient parent compared 3 key chromosome segment substitution lines and a plurality of positioning QTL containing (N29, N127 and N132) from mature seeds of gelatinization of starch It reduces the temperature of the endothermic peak forward, the results of the RVA spectra results. The corresponding characteristic values of starch DSC data show that the peak temperature, their corresponding end temperature, enthalpy, compared with the receptor parent decreased, close to the donor parent 9311. starch fine structure research found, containing from 9311 substitution segments N29, N127 and N132, the rice starch contains less A and B short chain chain, reduce speculation short branch amylopectin A chain and B1 chain content may be caused by the QTL effect. The comprehensive analysis of rice starch 3 extreme families may be due to contain less A chain and short chain B, more of the long chain group, jointly suppressed the swelling of starch granules, which leads to the changes of the physicochemical properties of.3 rice, genetic analysis of rice SSI gene alleles of the soluble starch synthase gene SSI in rice quality formation and the role of small, are minor base Because. But the exact effect has not been analyzed in detail. This study used with different SSI alleles (indica, japonica) source material and SSI-RNAi near isogenic lines, the expression of different SSI alleles, the physico-chemical quality and starch structure effect were analyzed. The results show that Xiangxi SSI, in between Indica and japonica two major alleles (SSI J and SSIi) expression differences caused a subtle change in the structure of rice starch, which showed a different effect on the physico-chemical quality. Rice varieties with different alleles of SSI were inhibited after SSI gene, the Physicochemical Quality of rice the significant effects of.SSI-RNAi transgenic plants significantly increased the amylose content, peak viscosity, breakdown value was significantly reduced. SSI allele genetic background, SSI-RNAi material performance are also different. SSI containing J allele Japonica background, RNAi rice setback than untransformed parental high significant differences; and in the SSI I allele of Indica Rice under the background of RNAi setback to the peak time of.RVA spectrum much lower than the parent reached extremely significant difference in indica rice RNAi lines and their parents, there was no difference in Japonica background. It also suggests that the effects of different SSI alleles on rice quality genetic is different. The rice starch DSC analysis showed that the SSI-RNAi material with untransformed parental compared to the initial temperature decrease is provided before gelatinization, heat value of Han descent. Starch crystallinity data show that RNAi system with respect to parents, the starch crystallinity decreased. Inhibition of expression of SSI gene can cause the change of starch crystal. It is suggested that expression of SSI gene in RNAi decreased, may cause the decrease of crystallinity of starch, the final The influence of physical and chemical quality and palatability of rice value. The results of that analysis and synthesis of SSIj allele might be responsible for the long chain of starch by GPC, once the interference in the structure of starch chain length were reduced, SSIi is likely responsible for the synthesis of low molecular weight chain. In transgenic lines can be seen when SSIi was inhibited after low molecular weight starch components decrease. By using near isogenic lines, we found that in the same Nipponbare (NIP) genetic background, different SSI alleles (SSIj and SSIi). The expression differences between the recurrent parent (NIP-SSIj) and its near isogenic line (the amylose content of rice NIP-SSIi close, near isogenic line NIP-NIL-SSIi rice RVA profile than the recurrent parent, found significantly increased the peak viscosity, hot paste viscosity decreased significantly, the breakdown value increased significantly. This also implies that the difference between Indica and Japonica Rice Quality in Wx major gene effects, and SS The variation of I alleles are related to.DSC analysis shows that the variation of SSI alleles leads to the onset temperature decreased significantly, showed early gelatinization, and peak viscosity, stop time, heat Han value compared with the recurrent parent, were not change crystalline variation of a certain degree of decline of.SSI allele caused by starch the degree. The results showed that when the SSIi import Longtefu in Nipponbare, the expression of SSI in near isogenic lines of genes changed, the consequent increase of low molecular weight short chain. This means that the SSI allele distribution function in Indica and japonica rice starch chain length differences, at the same time that a variation of SSI alleles resulted between Indica and Japonica Rice amylopectin structure differences. Based on the above research and the results obtained by a chromosome segment substitution lines in Japonica genetic background in two years under the environment The 14 parameters that affect the physico-chemical quality of the QTL analysis, a total of 35 QTL, of which 5 loci with pleiotropy; starch structure of 3 important families with multiple QTL positioning are analyzed, it provides a good reference for differences in rice quality analysis CSSL; two is the RNAi effect using the transgenic lines and near isogenic lines further in-depth analysis of rice SSI gene in two main allelic variation on rice quality and starch structure.

【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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