干扰水稻PULL和ISA3基因的表达对稻米品质影响的研究

发布时间：2018-06-09 16:43

  本文选题：水稻 + PULL基因　； 参考：《扬州大学》2016年硕士论文

【摘要】：淀粉是稻米胚乳的主要成分,在一定程度上决定了稻米的品质,淀粉合成是通过多种酶协同催化完成,其中淀粉去分支酶(Starch debranching enzyme,DBE)对淀粉最终结构的形成起着非常重要的作用,因此,对DBE的深入研究有助于解释淀粉生物合成机理。淀粉去分支酶包括普鲁兰酶(PULL)和异淀粉酶(ISA),异淀粉酶又包括ISA1、ISA2、ISA3三种同工型。本研究利用根癌农杆菌介导的转基因技术将PULL基因RNA干扰结构和ISA3基因RNA干扰结构导入受体品种日本晴中,且从2类干扰材料的杂交后代中通过PCR筛选到不同转化子来源的双基因干扰纯系。本实验中所用到的材料有的PULL和ISA3双干扰株系(PULL/ISA3-RNAi)四个,PULL干扰株系(PULL-RNAi)4个,ISA3干扰株系(ISA3-RNAi)3个和一个未转化对照。对获得的双干扰纯系和其亲本,以及未转化对照进行表达量分析,并测定稻米淀粉的理化特性和结构特性,以研究PULL、ISA3基因对稻米品质的影响。主要研究结果如下：(1)对双干扰材料及相关亲本水稻胚乳总RNA的Real-time PCR分析结果表明,单基因干扰材料中,PULL或ISA3基因的表达量不同程度的降低,且达到极显著水平。双基因干扰材料中PULL和ISA3的表达量与其对应的单基因干扰亲本材料相比无显著性差异。表明RNAi结构均已经发挥了作用,成功的抑制了相关基因的表达。(2)理化分析表明尸ULL/ISA3-RNAi,PULL-RNAi和ISA3-RNAi材料的染色范围和染色程度无明显的差别。转基因植株胚乳总淀粉含量、直链淀粉含量和可溶性总糖含量和未转化对照无明显差异,表明PULL或ISA3基因的低表达没有改变胚乳中淀粉分布和植物糖原分布。(3)对转基因材料的稻米淀粉RVA测定和DSC测定结果显示,转基因材料RVA和DSC的特征值与对照相比没有达到显著性差异。(4)对转基因材料的稻米淀粉进行X-射线衍射和红外光谱全反射分析,结果表明PULL/ISA3-RNAi,PULL-RNAi和ISA3-RNAi材料淀粉粒的晶型和结晶度无明显变化。(5)转基因株系稻米淀粉精细结构分析表明,PULL-RNAi和ISA3-RNAi株系中10≤DP≤20的淀粉链较未转化对照略微减少,大部分PULL/ISA3-RNAi材料胚乳中支链淀粉中10≤DP≤35的淀粉链均比单基因干扰材料含量高,说明PULL,ISA3都影响了支链淀粉的精细结构。(6)对转基因水稻农艺性状调查发现,PULL/ISA3-RNAi材料和单基因干扰材料的有效分蘖数、主穗长、结实率整体上和未转化对照无明显差异,植株株高和结实率也没有明显的规律性变化。
[Abstract]:Starch is the main component of rice endosperm, which determines the quality of rice to a certain extent. Starch synthesis is catalyzed by a variety of enzymes, among which starch debranching enzyme DBE plays a very important role in the formation of starch final structure. Therefore, the in-depth study of DBE is helpful to explain the mechanism of starch biosynthesis. Starch debranching enzymes include PULL) and isoamylase ISAA, and isoamylase include ISA1, ISA2 and ISA3 isoforms. In this study, the RNA interference structure of call gene and ISA3 gene RNA interference structure were introduced into the recipient variety Nippon by Agrobacterium tumefaciens mediated transgenic technology. Double gene interference pure lines from different transformants were screened by PCR from hybrid progenies of two kinds of interference materials. The materials used in this experiment were pul and ISA3 double interference lines (PULL / ISA3-RNAi), PULL / ISA3-RNAi4 (PULL-RNAi3), ISA3-RNAi3 (3) and one untransformed control. In order to study the effect of PULLN ISA3 gene on rice quality, the expression of double interference pure lines, their parents and untransformed control were analyzed, and the physicochemical and structural characteristics of rice starch were determined. The main results were as follows: (1) Real-time PCR analysis of total endosperm RNA of two interfering materials and their related parents showed that the expression of pail or ISA3 gene in single gene interference materials decreased in varying degrees and reached an extremely significant level. There was no significant difference in the expression of pul and ISA3 between the two gene interference materials and their monogenic interference parents. The results of physicochemical analysis showed that there was no significant difference in staining range and staining degree between PULL / ISA3-RNAi and ISA3-RNAi in PULL-RNAi and ISA3-RNAi. There was no significant difference in total starch content, amylose content and soluble sugar content between transgenic plants and untransformed control. The results showed that the low expression of pul or ISA3 gene did not change the starch distribution in endosperm and the distribution of plant glycogen. The characteristic values of RVA and DSC were not significantly different from those of the control. The results showed that the grain shape and crystallinity of starch granules of PULL / ISA3-RNAi and ISA3-RNAi were not significantly changed. The results of starch fine structure analysis showed that the starch chains of PULL-RNAi and ISA3-RNAi lines with 10 鈮，

本文编号：2000414