玉米NDH复合体核基因进化分析和NdhL亚基超表达水稻的耐盐性光合机制研究
发布时间：2018-08-17 16:35
【摘要】：随着盐碱地面积逐渐增大,盐胁迫已成为制约作物产量的一个重要的非生物胁迫因素。因此,不断探究作物耐盐的生理机制以提高粮食的产量和品质是人类不断追求的目标。高等植物NDH复合体介导的围绕PSI的循环电子传递(CET-PSI)是植物适应各种自然环境胁迫所产生的一个十分重要的保护机制。由于C4植物CO2浓缩机制需要额外ATP,其CET能力强于C3植物。NDH的表达变化是造成C4植物和C3植物CET强弱差异的主要因素。在三大粮食作物中,玉米和水稻分别属于C4和C3植物。本研究基于生物信息学方法全基因组鉴定玉米NDH复合体核编码基因,对鉴定出的基因基于基因家族分别开展系统进化分析,筛选C3和C4植物中可能发生功能分化的基因,并对玉米中筛选到的经历正选择作用的基因家族中的ZmNdhL1基因,在水稻中进行转基因超表达研究,比较分析转基因株系与野生型植株在盐胁迫下光合生理特性差异,主要结果如下:1、以17个NDH复合体核编码的亚基蛋白结构域为查询序列,搜索玉米、拟南芥、水稻和高粱参考基因组,共鉴定出27个玉米基因,22个拟南芥基因,23个水稻基因和23个高粱基因。依据基因重复与树图将上述NDH核基因的进化树分为三类。A类:四个物种构建出的树图为单簇且在四个物种中的基因均为单拷贝基因,此类进化树有8个;B类:四个物种构建的树图都为单簇,但在C4作物玉米或高粱中的基因有两个拷贝,此类进化树有5个;C类:四个物种构建的树图有两个以上的同源基因簇,此类进化树有4个。经正选择作用检测发现,NdhL和PnsB2两个基因家族经历了正选择作用。2、对玉米中NdhL和PnsB2两个基因家族的基因实时荧光定量分析发现,ZmNdhL1表达量最高,该基因在叶中高表达,受盐、干旱、低温非生物胁迫诱导表达。从具有强CET活性的玉米自交系ZM395中克隆了该基因的全长cDNA,其开放阅读框(ORF)为588bp,编码195个氨基酸。构建了该基因过表达载体,利用农杆菌介导法转化水稻,经过潮霉素抗性鉴定、PCR和RT-PCR鉴定筛选出24个转基因植株,其中转基因植株OE3和OE13表达最高,被用于后续研究。3、测定转基因和野生型植株叶片卷曲程度和叶绿素荧光动力学曲线发现,在对照情况下,超表达株系与野生型之间差异不明显。在200mM盐处理条件下,与野生型相比,超表达株系叶片卷曲程度低;作用光关闭后叶绿素荧光瞬间上升幅度大;PSⅠ的最大光化学效率Y(Ⅰ)、PSⅡ的最大光化学效率Y(Ⅱ)、PSⅠ电子传递速率ETRⅠ、PSⅡ电子传递速率ETRⅡ下降幅度小;快速叶绿素荧光OJIP曲线J点下移,K-band峰值小;延迟荧光诱导曲线的I1点和I2点下降幅度小。上述结果表明,超表达株系的耐盐性优于野生型。ZmNdhL1基因的过表达提高了植株的CET活性,使得光合电子传递链的放氧复合体(OEC)和电子由原初醌受体QA向下游传递的活性受伤害程度变轻,从而降低了盐胁迫对PSⅠ和PSⅡ的伤害。
[Abstract]:With the increasing of saline-alkali land area, salt stress has become an important abiotic stress factor restricting crop yield. Therefore, exploring the physiological mechanism of crop salt tolerance in order to improve grain yield and quality is the goal of human being. NDH complex mediated cyclic electron transfer (CET-PSI) around PSI in higher plants is an important protective mechanism for plants to adapt to various natural environment stresses. Because the mechanism of CO2 concentration in C4 plants requires extra ATP, the CET ability of C4 plants is stronger than that of C3 plants. The change of CET expression in C4 plants and C3 plants is the main factor causing the difference of CET intensity between C4 plants and C3 plants. Corn and rice belong to C _ 4 and C _ 3 respectively among the three main food crops. In this study, the nucleocoding genes of maize NDH complex were identified by bioinformatics method. Phylogenetic analysis was carried out based on gene families to screen the genes that might differentiate function in C3 and C4 plants. The ZmNdhL1 genes in the gene family of maize which experienced positive selection were studied by transgenic superexpression in rice, and the differences of photosynthetic physiological characteristics between transgenic lines and wild type plants under salt stress were compared and analyzed. The main results were as follows: 1. The reference genomes of maize, Arabidopsis, rice and sorghum were searched using 17 subunit protein domains encoded by NDH complex as query sequences. Twenty-seven maize genes, 22 Arabidopsis genes, 23 rice genes and 23 sorghum genes were identified. According to the gene duplication and tree map, the evolutionary tree of the above NDH nuclear gene is divided into three categories: the tree map constructed by four species is a single cluster and the genes in each of the four species are single copy genes. The phylogenetic tree has eight species B: four species construct a single cluster, but there are two copies of genes in C4 crop corn or sorghum, and the evolutionary tree has five C groups: four species construct a tree map with more than two homologous gene clusters. There are 4 such evolutionary trees. The positive selection test showed that the two gene families of NdhL and PnsB2 experienced positive selection. The results of real-time fluorescence quantitative analysis of NdhL and PnsB2 genes in maize showed that ZmNdhL1 expressed the highest amount in the leaves, and the gene was highly expressed, salt and drought. Low temperature abiotic stress induces expression. The full-length cDNA of this gene was cloned from maize inbred line ZM395 with strong CET activity. The open reading frame (ORF) is 588bpand encodes 195 amino acids. The overexpression vector was constructed and transformed into rice by Agrobacterium tumefaciens. 24 transgenic plants were screened by hygromycin resistance identification and RT-PCR identification, among which the OE3 and OE13 expression of transgenic plants was the highest. The leaf curl degree and chlorophyll fluorescence kinetic curves of transgenic and wild type plants were determined. The results showed that there was no significant difference between the overexpressed lines and wild type in the control. Under the condition of 200mM salt treatment, the leaf curl degree of the overexpression strain was lower than that of wild type. The maximum photochemical efficiency Y (鈪，
本文编号：2188245
[Abstract]:With the increasing of saline-alkali land area, salt stress has become an important abiotic stress factor restricting crop yield. Therefore, exploring the physiological mechanism of crop salt tolerance in order to improve grain yield and quality is the goal of human being. NDH complex mediated cyclic electron transfer (CET-PSI) around PSI in higher plants is an important protective mechanism for plants to adapt to various natural environment stresses. Because the mechanism of CO2 concentration in C4 plants requires extra ATP, the CET ability of C4 plants is stronger than that of C3 plants. The change of CET expression in C4 plants and C3 plants is the main factor causing the difference of CET intensity between C4 plants and C3 plants. Corn and rice belong to C _ 4 and C _ 3 respectively among the three main food crops. In this study, the nucleocoding genes of maize NDH complex were identified by bioinformatics method. Phylogenetic analysis was carried out based on gene families to screen the genes that might differentiate function in C3 and C4 plants. The ZmNdhL1 genes in the gene family of maize which experienced positive selection were studied by transgenic superexpression in rice, and the differences of photosynthetic physiological characteristics between transgenic lines and wild type plants under salt stress were compared and analyzed. The main results were as follows: 1. The reference genomes of maize, Arabidopsis, rice and sorghum were searched using 17 subunit protein domains encoded by NDH complex as query sequences. Twenty-seven maize genes, 22 Arabidopsis genes, 23 rice genes and 23 sorghum genes were identified. According to the gene duplication and tree map, the evolutionary tree of the above NDH nuclear gene is divided into three categories: the tree map constructed by four species is a single cluster and the genes in each of the four species are single copy genes. The phylogenetic tree has eight species B: four species construct a single cluster, but there are two copies of genes in C4 crop corn or sorghum, and the evolutionary tree has five C groups: four species construct a tree map with more than two homologous gene clusters. There are 4 such evolutionary trees. The positive selection test showed that the two gene families of NdhL and PnsB2 experienced positive selection. The results of real-time fluorescence quantitative analysis of NdhL and PnsB2 genes in maize showed that ZmNdhL1 expressed the highest amount in the leaves, and the gene was highly expressed, salt and drought. Low temperature abiotic stress induces expression. The full-length cDNA of this gene was cloned from maize inbred line ZM395 with strong CET activity. The open reading frame (ORF) is 588bpand encodes 195 amino acids. The overexpression vector was constructed and transformed into rice by Agrobacterium tumefaciens. 24 transgenic plants were screened by hygromycin resistance identification and RT-PCR identification, among which the OE3 and OE13 expression of transgenic plants was the highest. The leaf curl degree and chlorophyll fluorescence kinetic curves of transgenic and wild type plants were determined. The results showed that there was no significant difference between the overexpressed lines and wild type in the control. Under the condition of 200mM salt treatment, the leaf curl degree of the overexpression strain was lower than that of wild type. The maximum photochemical efficiency Y (鈪，
本文编号：2188245
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