本文關(guān)鍵詞： 大豆 同源異型域亮氨酸拉鏈蛋白(HD-Zip) 轉(zhuǎn)錄因子 百脈根 耐鹽性　出處：《中國(guó)農(nóng)業(yè)科學(xué)》2017年09期 　論文類型：期刊論文

【摘要】：【目的】從大豆鹽脅迫基因表達(dá)譜中篩選并克隆得到同源異型域亮氨酸拉鏈蛋白(HD-Zip)家族基因GmHAT5,將其轉(zhuǎn)化豆科模式植物百脈根并進(jìn)一步探究其抗鹽調(diào)控機(jī)制�！痉椒ā渴褂枚喾N生物信息學(xué)軟件對(duì)GmHAT5的開放讀碼框(ORF)、編碼蛋白的分子量、等電點(diǎn)、序列結(jié)構(gòu)和蛋白定位等進(jìn)行預(yù)測(cè),同時(shí)將大豆GmHAT5蛋白與其他10個(gè)物種的同源蛋白進(jìn)行比對(duì)分析,并對(duì)GmHAT5在大豆不同器官及鹽脅迫下的表達(dá)特性進(jìn)行分析。此外,構(gòu)建GmHAT5的植物超表達(dá)載體,通過(guò)對(duì)發(fā)根農(nóng)桿菌LBA1334的轉(zhuǎn)化,得到"復(fù)合體"百脈根植株,并在鹽脅迫條件下對(duì)其進(jìn)行抗鹽表型分析;通過(guò)對(duì)根癌農(nóng)桿菌EHA105的轉(zhuǎn)化,獲得百脈根的穩(wěn)定轉(zhuǎn)化株系,并對(duì)其進(jìn)行抗鹽表型分析及相關(guān)生理指標(biāo)檢測(cè)�！窘Y(jié)果】多種生物信息學(xué)軟件分析表明,該片段包含1個(gè)1 038 bp的ORF,編碼345個(gè)氨基酸。GmHAT5的理論分子量和等電點(diǎn)分別為39.17 k D和4.63。GmHAT5蛋白定位于細(xì)胞核,與其他HD-Zip家族蛋白一樣,屬于典型的核蛋白。序列分析表明,GmHAT5包含一個(gè)同源異型結(jié)構(gòu)域和一個(gè)亮氨酸拉鏈結(jié)構(gòu)域,屬于第I類同源異型域亮氨酸拉鏈蛋白。同源蛋白比對(duì)表明其與野生大豆GsHAT5同源性最高�；虮磉_(dá)特性分析表明,GmHAT5在大豆植株的各個(gè)不同器官中均有表達(dá),是一個(gè)受鹽誘導(dǎo)上調(diào)表達(dá)的HD-Zip類轉(zhuǎn)錄因子。發(fā)狀根轉(zhuǎn)化的結(jié)果表明,使用200 mmol·L~(-1) NaCl處理植株7 d后,"復(fù)合體"植株生長(zhǎng)狀態(tài)良好,對(duì)照組植株葉片明顯萎蔫、失綠;不同NaCl濃度處理離體轉(zhuǎn)基因發(fā)狀根14 d后,對(duì)照組較試驗(yàn)組發(fā)狀根明顯干枯、生長(zhǎng)受到抑制。穩(wěn)定轉(zhuǎn)化結(jié)果顯示,不同鹽濃度處理14 d后,轉(zhuǎn)GmHAT5百脈根與兩組對(duì)照植株相比生長(zhǎng)狀態(tài)更好。相關(guān)生理指標(biāo)檢測(cè)結(jié)果顯示,與兩組對(duì)照相比,轉(zhuǎn)基因百脈根植株中丙二醛含量和相對(duì)質(zhì)膜透性明顯降低(P0.05),而葉綠素含量和根系活力則顯著升高(P0.05)。測(cè)定不同植株陽(yáng)離子含量的結(jié)果表明,轉(zhuǎn)基因百脈根株系與兩組對(duì)照相比,Na~+在葉片和根中含量顯著下降,K~+和Ca~(2+)在葉片和根中含量顯著升高�！窘Y(jié)論】從大豆中克隆得到一個(gè)編碼HD-ZipⅠ類同源異型域亮氨酸拉鏈蛋白基因GmHAT5,該基因在大豆中受鹽脅迫誘導(dǎo)表達(dá)量顯著升高。超量表達(dá)GmHAT5顯著增強(qiáng)百脈根的耐鹽能力,發(fā)狀根轉(zhuǎn)化法可以作為一種快速有效篩選抗鹽候選基因的手段。推測(cè)GmHAT5在大豆鹽脅迫應(yīng)激調(diào)控中扮演重要角色。
[Abstract]:[objective] to screen and clone the homologous gene GmHAT5 of leucine zipper HD-Zipprotein family from soybean salt stress gene expression profile, to transform GmHAT5 into the legume model plant, and to further explore its salt-resistance regulation mechanism. Using a variety of bioinformatics software, the open reading frame of GmHAT5, encoding the molecular weight of the protein, The isoelectric point, sequence structure and protein location were predicted. At the same time, the soybean GmHAT5 protein was compared with the homologous proteins of other 10 species, and the expression characteristics of GmHAT5 in different organs and salt stress of soybean were analyzed. The plant superexpression vector of GmHAT5 was constructed. By transforming LBA1334 of Agrobacterium tumefaciens, the plant of "complex" was obtained, and the salt-resistant phenotype was analyzed under salt stress, and the transformation of EHA105 from Agrobacterium tumefaciens to Agrobacterium tumefaciens was carried out. The stable transformation lines were obtained, and the salt-resistant phenotypic analysis and physiological index detection were carried out. [results] many bioinformatics software analysis showed that, The fragment contains 1 038 BP ORF and encodes 345 amino acids. GmHAT5 has a theoretical molecular weight of 39.17 KD and a isoelectric point of 4.63 GmHAT5, which are located in the nucleus, just like other HD-Zip family proteins. GmHAT5 contains a homologous domain and a leucine zipper domain. Leucine zipper protein belongs to class I homologous domain. Homologous protein comparison shows that it has the highest homology with wild soybean GsHAT5. The analysis of gene expression characteristics shows that GmHAT5 is expressed in different organs of soybean plant. The results of hairy root transformation showed that after treated with 200 mmol 路L ~ (-1) NaCl for 7 days, the "complex" plants grew well, and the leaves of the control plants were wilted and green. After treated with different concentrations of NaCl for 14 days, the hair roots of the control group were significantly dry and the growth was inhibited compared with the experimental group. The stable transformation results showed that the control group was treated with different concentrations of salt for 14 days. Compared with the control group, the growth status of the GmHAT5 100 vein root was better than that of the two groups, and the relative physiological indexes showed that, compared with the two control groups, the plant growth status was better than that of the control group. The content of malondialdehyde and relative plasma membrane permeability of the transgenic plants decreased significantly, while the chlorophyll content and root activity increased significantly. The results showed that the contents of cations in different plants were determined. Compared with the two groups, the content of Na ~ + in leaves and roots of transgenic Baimai root lines decreased significantly. The contents of K ~ and Ca~(2) in leaves and roots increased significantly. [conclusion] A homologous domain Leucine encoding HD-Zip class I was cloned from soybean. The acid zipper protein gene GmHAT5, which was induced to express in soybean under salt stress, was significantly increased. Overexpression of GmHAT5 significantly enhanced the salt-tolerance ability of the root. Hairy root transformation can be used as a rapid and effective method to screen candidate genes for salt resistance. It is speculated that GmHAT5 plays an important role in the regulation of salt stress in soybean.
【作者單位】： 信陽(yáng)師范學(xué)院生命科學(xué)學(xué)院/大別山農(nóng)業(yè)生物資源保護(hù)與利用研究院;
【基金】：國(guó)家自然科學(xué)基金青年基金(31400213) 信陽(yáng)師范學(xué)院青年骨干教師資助計(jì)劃(2015);信陽(yáng)師范學(xué)院“南湖學(xué)者獎(jiǎng)勵(lì)計(jì)劃”青年項(xiàng)目 2016年國(guó)家級(jí)大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃(201610477011)
【分類號(hào)】：S541.6

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