水稻矮化剑叶卷曲基因DCFL1的图位克隆和功能研究

发布时间：2019-06-21 21:18

【摘要】：株高是水稻最重要的农艺性状之一,直接影响水稻的抗倒伏和产量,大量矮杆突变体的出现引发了“第二次绿色革命”。剑叶是水稻最重要的功能叶,其形态特征关系到叶片间的通透性和光能利用率,直接影响水稻灌浆速度和籽粒饱满度。因此,了解水稻株高的遗传模式和剑叶发育的分子机理对水稻株型改良和丰产具有极其重要的意义。利用EMS诱变籼型水稻恢复系缙恢10号,从其后代鉴定到一个矮化剑叶基部特异卷曲突变体,暂命名为dcfl1(dwarf and curled flag leaf 1)。本文对dcfl1进行了表型和细胞学观察,光合色素含量和光合参数分析,并开展了基因定位、图位克隆、表达模式分析和功能验证等研究。主要结果如下:1.dcfl1的表型分析和农艺性状调查突变体dcfl1全生育期表现出矮化性状,dcfl1的细胞长度明显比野生型短,达到了极显著水平,而细胞宽度无显著差异。抽穗期dcfl1剑叶的叶片和叶鞘连接处硬化,剑叶基部展开受阻,半边叶片向内卷曲。剑叶上部和中部正常,其他叶片也正常。农艺性状分析发现,与野生型相比,dcfl1的有效穗数为14.2,极显著高于野生型的11.6,穗粒数、实粒数、结实率和千粒重等则无显著变化。2.dcfl1的光合色素含量和光合参数测定开花期对光合色素含量测定结果显示,dcfl1的剑叶、倒2叶和倒3叶的叶绿素a含量均极显著高于野生型,类胡萝卜素含量也略有升高,但仅剑叶达到极显著差异水平,叶绿素b含量则无显著变化。光合参数测定发现,与野生型相比,dcfl1的胞间CO2浓度无明显变化,净光合速率Pn、气孔导度Gs和蒸腾速率Tr则显著降低。3.dcfl1的遗传分析与基因定位利用杂交组合西农1A/dcfl1的F1、F2群体进行遗传分析,表明dcfl1突变表型受1对隐性核基因控制。以西农1A/dcfl1杂交组合的620株F2隐性单株为定位群体,利用SSR标记和In Del标记进行基因定位,最终将DCFL1定位在第3染色体Ind03-11和Ind03-6之间,物理距离约为78kb。4.DCFL1基因的克隆与蛋白分析对定位区间内的15个注释基因进行基因测序,发现LOC_Os03g04680在dcfl1中发生了一个G-A的碱基替换,导致编码的氨基酸从半胱氨酸变成了酪氨酸,初步确定为DCFL1的候选基因。DCFL1编码细胞色素P450加单氧酶Os CYP96B4,属于CYP86家族中的CYP96亚家族。5.DCFL1候选基因的确定利用野生型DCFL1基因组片段包括上游2875bp、编码框和下游1315bp构建互补表达载体,通过农杆菌介导遗传转化突变体dcfl1。在转基因植株中,我们共鉴定了12株阳性转基因植株,表型观察发现突变体表型得以恢复,确定了LOC_Os03g04680是DCFL1基因。6.DCFL1基因的表达模式分析通过半定量RT-PCR和q PCR等技术进行表达模式分析,结果表明DCFL1在根、茎、叶和穗中都有表达;在叶片和叶鞘中表达量较高,其中,剑叶叶鞘表达量最高。利用GUS试验进一步探究DCFL1基因表达模式。GUS染色结果表明DCFL1基因在各器官均有表达,这与半定量RT-PCR和q PCR结果一致。7.DCFL1基因与激素的关系激素处理实验发现不同浓度的BR和GA3均无法使突变体dcfl1和等位突变体dcfl1-1矮化表型得以恢复。说明两个突变体的矮化表型不受外源BR和GA3的影响,但在没有验证其他植物激素的响应以及没有检测突变体dcfl1和突变体dcfl1-1内源激素的前提下,不能排除DCFL1参与植物激素代谢途径的可能性。8.DCFL1基因可能参与表皮蜡质的合成对突变体dcfl1卷曲的剑叶基部表皮进行扫描电镜观察发现dcfl1的两个小叶脉之间存在沟壑,导致dcfl1表皮蜡质覆盖面积减少。叶绿素浸析试验结果发现dcfl1的表皮渗透性升高,导致叶绿素比野生型更容易提取出来,暗示着dcfl1表皮蜡质含量要低于野生型。这些结果表明DCFL1可能参与表皮蜡质的合成。
[Abstract]:The plant height is one of the most important agronomic characters of rice, which directly affects the lodging and yield of rice, and the emergence of a large number of short-stem mutants has raised the "the second green revolution". Swordleaf is one of the most important functional leaves of rice, and its morphological characteristics are related to the permeability and light energy utilization of the leaves, which directly affects the grain filling speed and the fullness of the grain. Therefore, it is of great significance to understand the high genetic pattern of rice plant and the molecular mechanism of the development of swordleaf. A dwarf sword leaf base specific curl mutant was identified from the progeny of the rice restorer line of the rice-type rice restorer line of the EMS by using the EMS, and it was temporarily named dcfl1 (dwarf and curled flag leaf 1). In this paper, the phenotype and cytological observation of dcfl1, the content of photosynthetic pigment and the analysis of the photosynthetic parameters were carried out, and the research of gene location, site cloning, expression pattern analysis and functional verification was carried out. The main results are as follows:1. The phenotype and agronomic characters of dcfl1 show that the whole growth period of the mutant dcfl1 shows the dwarfing character, and the cell length of dcfl1 is significantly shorter than that of the wild type, and the cell width is not significantly different. At the junction of the blade and the blade of the dcfl1 swordleaf of the heading stage, the base of the blade was blocked, and the half of the blade was curled inward. The upper part and the middle part of the blade are normal, and the other blades are normal. The results showed that, compared with wild-type, the effective spikelet number of dcfl1 was 14.2, which was significantly higher than that of the wild-type 11.6, the number of kernels per spike, the number of grains per ear, the seed setting and the 1000-grain weight were not changed significantly. The content of chlorophyll a was significantly higher than that of wild type, but the content of carotenoid increased slightly, but the content of chlorophyll b did not change significantly. The results showed that there was no significant change in the intercell CO2 concentration of dcfl1, the net photosynthetic rate (Pn), the stomatal conductance (Gs) and the transpiration rate (Tr) decreased significantly in comparison with the wild type. It is shown that the dcfl1 mutant phenotype is controlled by 1 recessive nuclear gene. The 620 strain F2 recessive single plant of the hybrid combination of the No. 1A/ dcfl1 west of the west is a positioning group, and the gene is positioned by using the SSR marker and the In Del marker, and the DCFL1 is finally positioned between the third chromosome In03-11 and the In03-6, The physical distance was about 78 kb.4. The cloning and protein analysis of the DCFL1 gene were used to sequence the 15 annotation genes in the localization interval, and found that LOC _ Os03g04680 had a G-A base substitution in dcfl1, resulting in the encoded amino acid being transformed from cysteine into tyrosine and preliminarily determined as the candidate gene of DCFL1. DCFL1 encodes a cytochrome P450-derived CYP96B4, which belongs to the CYP96 subfamily of the CYP86 family. In the transgenic plant, we identified 12 positive transgenic plants and found that the phenotype of the mutant was restored. The expression pattern of the DCFL1 gene was analyzed by semi-quantitative RT-PCR and q-PCR. The results showed that DCFL1 was in the root and the stem. The expression of leaf and spikelet was high, and the expression of leaf and leaf was the highest. The expression pattern of DCFL1 gene was further explored by GUS test. The results of GUS staining showed that the DCFL1 gene was expressed in all organs, which was consistent with the results of semi-quantitative RT-PCR and q-PCR. The dwarfing phenotype of the two mutants is not affected by the foreign BR and the GA3, but on the premise of not verifying the response of other plant hormones and the absence of the endogenous hormones of the mutant dcfl1 and the mutant dcfl1-1, The possibility of DCFL1 in the pathway of plant hormone metabolism could not be ruled out. The results of the chlorophyll-leaching test showed that the increase of the skin permeability of dcfl1 resulted in a much easier extraction of the chlorophyll than the wild-type, suggesting that the waxy content of the dcfl1 was lower than that of the wild-type. These results suggest that DCFL1 may be involved in the synthesis of the skin wax.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511

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