【摘要】：小麥是我國重要的糧食作物。小麥的產量由畝穗數(shù)、穗粒數(shù)和千粒重決定,分蘗數(shù)在一定程度上決定了最終的畝穗數(shù)。目前對小麥分蘗的研究大多集中在生理方面,對其發(fā)生的分子機理及調控方面的研究國內外報道很少。已有研究表明獨角金內酯(SLs)作為一種植物激素可以抑制水稻和擬南芥?zhèn)戎Φ纳L。DWARF27(D27)基因編碼一種含鐵的β類胡蘿卜素異構酶,參與SLs的合成。d27突變體表現(xiàn)為側枝增多,株高下降,株型緊湊。為理解SLs調控小麥分蘗的分子機理,本研究分離了TaDWARF27基因,并對其表達模式及功能進行了初步研究,具體結果如下:TaDWARF27基因具有三對等位基因,定位于7號染色體上。三對等位基因的序列同源性高達97.71%。cDNAs全長分別為837bp(TaD27-7A)、840bp(TaD27-7B)和840bp(TaD27-7D),分別編碼279、280、280個氨基酸殘基組成的蛋白質。該基因gDNA具有7個外顯子和6個內含子,全長分別為:4675bp(TaD27-7A)、4896bp(TaD27-7B)和4703bp(TaD27-7D)。與OsD27相似,TaD27蛋白具有一個保守的結構域DUF4033。系統(tǒng)進化樹分析表明TaD27蛋白與粗山羊草D27蛋白親緣關系最近,其次為大麥和二穗短柄草的D27蛋白。亞細胞定位分析顯示TaD27蛋白定位于葉綠體上。定量PCR分析結果顯示TaD27的轉錄物主要在葉片中表達。原位雜交顯示TaD27主要在葉片、葉腋和幼穗原基表達。TaD27-7B的過表達可以恢復擬南芥d27突變體的表型,這說明在擬南芥和小麥中TaD27的功能具有一定的保守性。為進一步探究TaD27的功能,我們構建了RNA interference載體和過表達載體,以栽培品種科農199(KN199)的幼胚為受體,進行農桿菌浸染介導的小麥遺傳轉化,共得到了TaD27 RNAi轉基因植株295株和過表達轉基因植株255株。除草劑抗性篩選和PCR分子鑒定得到16株RNAi陽性苗和10株過表達陽性苗。定量PCR分析結果顯示RNAi陽性苗葉片中TaD27的表達量明顯下調。我們從中選出13個RNAi株系和2個過表達株系進行了加代,目前正進行植株鑒定及表型觀察。綜上所述,TaD27很可能在小麥分蘗調控過程中起重要作用,該實驗結果可以為提高小麥產量提供分子理論依據。
[Abstract]:Wheat is an important food crop in China. The yield of wheat is determined by the number of ears per mu, the number of grains per ear and the weight of 1000 grains, and the number of tillers determines the number of ears per mu to a certain extent. At present, the studies on wheat tillers are mostly focused on physiology, and few reports on molecular mechanism and regulation of wheat tillers are reported at home and abroad. It has been shown that (SLs), as a phytohormone, can inhibit the growth of lateral branches of rice and Arabidopsis thaliana. DWARF27 (D27) gene encodes an iron-containing 尾 -carotene isomerase. D 27 mutants were characterized by increased lateral branches, decreased plant height and compact plant type. In order to understand the molecular mechanism of SLs regulating wheat tiller, TaDWARF27 gene was isolated, and its expression pattern and function were studied. The results are as follows: TaDWARF27 gene has three alleles located on chromosome 7. The sequence homology of the three alleles is as high as 837bp (TaD27-7A), 840bp (TaD27-7B) and 840bp (TaD27-7D), which encode 279280280 amino acid residues. There are 7 exons and 6 introns in gDNA, which are 4675bp (TaD27-7A), 4896bp (TaD27-7B) and 4703bp (TaD27-7D). Similar to OsD27, TaD27 protein has a conserved domain DUF4033. Phylogenetic tree analysis showed that TaD27 protein was most closely related to D27 protein of Leymus chinensis, followed by D27 protein from barley and short stalks. Subcellular localization analysis showed that TaD27 protein was located on chloroplast. Quantitative PCR analysis showed that TaD27 transcripts were mainly expressed in leaves. In situ hybridization showed that TaD27 was mainly expressed in leaf, axil and young panicle primordia. Overexpression of TaD27-7B could restore the phenotype of Arabidopsis d27 mutant, which indicated that the function of TaD27 was conserved in Arabidopsis and wheat. In order to further explore the function of TaD27, we constructed RNA interference vector and overexpression vector. The immature embryos of the cultivar KN199 were used as the receptor for Agrobacterium tumefaciens soaking mediated wheat genetic transformation. A total of 295 TaD27 RNAi transgenic plants and 255 overexpressed transgenic plants were obtained. 16 RNAi positive seedlings and 10 overexpression positive seedlings were obtained by herbicide resistance screening and PCR molecular identification. Quantitative PCR analysis showed that the expression of TaD27 in the leaves of RNAi positive seedlings was significantly down-regulated. 13 RNAi lines and 2 overexpression lines were selected for culture. Plant identification and phenotypic observation were carried out. In conclusion, TaD27 may play an important role in the regulation of wheat tillering, and the results can provide molecular theoretical basis for improving wheat yield.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S512.1;Q943.2

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1 武亭亭;小麥獨腳金內酯合成相關基因TaDWARF27的分離與功能分析[D];山東農業(yè)大學;2016年

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本文編號：2429863