【摘要】：隨著鹽堿地面積逐漸增大,鹽脅迫已成為制約作物產(chǎn)量的一個(gè)重要的非生物脅迫因素。因此,不斷探究作物耐鹽的生理機(jī)制以提高糧食的產(chǎn)量和品質(zhì)是人類不斷追求的目標(biāo)。高等植物NDH復(fù)合體介導(dǎo)的圍繞PSI的循環(huán)電子傳遞(CET-PSI)是植物適應(yīng)各種自然環(huán)境脅迫所產(chǎn)生的一個(gè)十分重要的保護(hù)機(jī)制。由于C4植物CO2濃縮機(jī)制需要額外ATP,其CET能力強(qiáng)于C3植物。NDH的表達(dá)變化是造成C4植物和C3植物CET強(qiáng)弱差異的主要因素。在三大糧食作物中,玉米和水稻分別屬于C4和C3植物。本研究基于生物信息學(xué)方法全基因組鑒定玉米NDH復(fù)合體核編碼基因,對(duì)鑒定出的基因基于基因家族分別開展系統(tǒng)進(jìn)化分析,篩選C3和C4植物中可能發(fā)生功能分化的基因,并對(duì)玉米中篩選到的經(jīng)歷正選擇作用的基因家族中的ZmNdhL1基因,在水稻中進(jìn)行轉(zhuǎn)基因超表達(dá)研究,比較分析轉(zhuǎn)基因株系與野生型植株在鹽脅迫下光合生理特性差異,主要結(jié)果如下:1、以17個(gè)NDH復(fù)合體核編碼的亞基蛋白結(jié)構(gòu)域?yàn)椴樵冃蛄?搜索玉米、擬南芥、水稻和高粱參考基因組,共鑒定出27個(gè)玉米基因,22個(gè)擬南芥基因,23個(gè)水稻基因和23個(gè)高粱基因。依據(jù)基因重復(fù)與樹圖將上述NDH核基因的進(jìn)化樹分為三類。A類:四個(gè)物種構(gòu)建出的樹圖為單簇且在四個(gè)物種中的基因均為單拷貝基因,此類進(jìn)化樹有8個(gè);B類:四個(gè)物種構(gòu)建的樹圖都為單簇,但在C4作物玉米或高粱中的基因有兩個(gè)拷貝,此類進(jìn)化樹有5個(gè);C類:四個(gè)物種構(gòu)建的樹圖有兩個(gè)以上的同源基因簇,此類進(jìn)化樹有4個(gè)。經(jīng)正選擇作用檢測(cè)發(fā)現(xiàn),NdhL和PnsB2兩個(gè)基因家族經(jīng)歷了正選擇作用。2、對(duì)玉米中NdhL和PnsB2兩個(gè)基因家族的基因?qū)崟r(shí)熒光定量分析發(fā)現(xiàn),ZmNdhL1表達(dá)量最高,該基因在葉中高表達(dá),受鹽、干旱、低溫非生物脅迫誘導(dǎo)表達(dá)。從具有強(qiáng)CET活性的玉米自交系ZM395中克隆了該基因的全長(zhǎng)cDNA,其開放閱讀框(ORF)為588bp,編碼195個(gè)氨基酸。構(gòu)建了該基因過(guò)表達(dá)載體,利用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化水稻,經(jīng)過(guò)潮霉素抗性鑒定、PCR和RT-PCR鑒定篩選出24個(gè)轉(zhuǎn)基因植株,其中轉(zhuǎn)基因植株OE3和OE13表達(dá)最高,被用于后續(xù)研究。3、測(cè)定轉(zhuǎn)基因和野生型植株葉片卷曲程度和葉綠素?zé)晒鈩?dòng)力學(xué)曲線發(fā)現(xiàn),在對(duì)照情況下,超表達(dá)株系與野生型之間差異不明顯。在200mM鹽處理?xiàng)l件下,與野生型相比,超表達(dá)株系葉片卷曲程度低;作用光關(guān)閉后葉綠素?zé)晒馑查g上升幅度大;PSⅠ的最大光化學(xué)效率Y(Ⅰ)、PSⅡ的最大光化學(xué)效率Y(Ⅱ)、PSⅠ電子傳遞速率ETRⅠ、PSⅡ電子傳遞速率ETRⅡ下降幅度小;快速葉綠素?zé)晒釵JIP曲線J點(diǎn)下移,K-band峰值小;延遲熒光誘導(dǎo)曲線的I1點(diǎn)和I2點(diǎn)下降幅度小。上述結(jié)果表明,超表達(dá)株系的耐鹽性優(yōu)于野生型。ZmNdhL1基因的過(guò)表達(dá)提高了植株的CET活性,使得光合電子傳遞鏈的放氧復(fù)合體(OEC)和電子由原初醌受體QA向下游傳遞的活性受傷害程度變輕,從而降低了鹽脅迫對(duì)PSⅠ和PSⅡ的傷害。
[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，

本文編號(hào)：2188245