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【目的】克隆酿酒酵母(Saccharomyces cerevisiae)耐盐基因HAL1(halotolerance)并转化棉花,探究该基因在棉花中的功能,进一步探究酵母耐盐相关基因在高等植物中的具体功能。【方法】根据随NCBI核酸数据库中酵母耐盐基因HAL1的mRNA全长序列信息,利用RT-PCR技术从酿酒酵母AS2.375中克隆该基因。选择酶切位点XbaⅠ和SmaⅠ对表达载体pBI121::GFP进行双酶切,采用In-Fusion技术构建pBI 121-ScHAL1::GFP融合表达载体。以自发荧光较弱的陆地棉品种Y-2067、ZA-23、GZ-2的花粉为材料,用基因枪轰击棉花花粉进行瞬时表达研究。采用基因枪活体转化技术将外源基因表达载体pBI121-HAL1::GFP转化棉花盐敏感材料中s9612,获得T_0棉花转基因种子。用100mol·L~(-1)NaCl盐溶液对转基因T_0种子进行耐盐性发芽试验,并进行分子检测,对鉴定出的转基因植株进行叶盘耐盐性分析。【结果】从酿酒酵母As2.375中克隆耐盐基因ScHAL1,基因全长885 bP,共编码294个氨基酸。对其序列进行分析,发现HAL1蛋白中丝氨酸所占比例最大,整个蛋白呈碱性且带正电,属于亲水性蛋白。根据蛋白二级结构预测结果,推测该蛋白的结构功能域可能主要由无规则卷曲和β-折叠片构成。棉花花粉瞬时表达结果表明,转化HAL1后,3种陆地棉花粉的绿色荧光现象都明显增强,说明该基因可以在这3种陆地棉的花粉中表达。用100 mol·L~(-1)NaCl盐溶液对转基因棉花T_0种子和受体材料中s9612自交种进行胁迫,发现转基因种子萌发能力明显强于受体材料,表明HAL1可以提高种子耐盐性。根据基因核苦酸序列设计2对引物对T_0转基因棉花幼苗进行分子检测,将纯化后的PCR产物进行直接测序,测序结果证明转基因成功。对鉴定出的转基因植株进行叶盘耐盐性分析发现;在600 mol·L~(-1)NaCl和400 mol·L~(-1)NaCl盐胁迫下转基因植株叶盘叶绿素含量均高于对照植株,且在600 mol·L~(-1)NaCl溶液胁迫后,转HAL1植株叶绿素含量反而高于400 mol·L~(-1)NaCl溶液处理后的叶盘。【结论】成功从酿酒酵母中克隆基因HAL1;酵母HAL1对提高棉花耐盐性具有重要作用。
【Objective】 The purpose of this study was to clone and transform the gene HAL1 (halotolerance) of Saccharomyces cerevisiae into cotton and explore the function of this gene in cotton and to further explore the function of yeast salt-tolerance-related genes in higher plants. 【Method】 The gene was cloned from Saccharomyces cerevisiae AS2.375 by RT-PCR based on the full-length sequence of HAL1, a yeast salt-tolerant gene in the NCBI database. The expression vector pBI121 :: GFP was double digested by XbaⅠ and SmaⅠ, and the pBI 121-ScHAL1 :: GFP fusion expression vector was constructed by In-Fusion technology. The spontaneous low fluorescence of upland cotton varieties Y-2067, ZA-23, GZ-2 pollen as material, using particle bombardment of cotton pollen transient expression studies. The gene-transfer vector pBI121-HAL1 :: GFP was transformed into s9612 cotton-sensitive material by using gene gun live transformation technique to obtain T_0 cotton transgenic seed. Salt-tolerant germination test of transgenic T 0 seed with 100 mol·L -1 NaCl salt solution was carried out, and the molecular test was carried out. The leaf disc salt tolerance of the identified transgenic plants was analyzed. 【Result】 ScHAL1 gene was cloned from Saccharomyces cerevisiae As2.375 and the full-length gene was 885 bP, encoding a total of 294 amino acids. Analysis of its sequence found that HAL1 protein serine in the largest proportion of the entire protein was alkaline and positively charged, is a hydrophilic protein. According to the predictions of protein secondary structure, it is speculated that the structural domain of the protein may be mainly composed of random curl and β-sheet. The transient expression of cotton pollen showed that after transformation of HAL1, the green fluorescence of the three upland cotton pollen significantly increased, indicating that the gene can be expressed in the pollens of these three kinds of upland cotton. Stressing on the s9612 selfing seeds of transgenic cotton T0 seed and recipient material with 100 mol·L ~ (-1) NaCl salt solution, the germination ability of the transgenic seeds was significantly stronger than that of the recipient material, indicating that HAL1 can improve the seed salt tolerance . According to the nucleotide sequence of the gene, two pairs of primers were designed to detect the molecular cotton seedlings of T_0 transgenic cotton. The purified PCR products were directly sequenced, and the sequencing results proved that the transgene was successful. The analysis of leaf salt tolerance of the identified transgenic plants showed that the leaf chlorophyll content of transgenic plants under salt stress of 600 mol·L -1 NaCl and 400 mol·L -1 NaCl was higher than that of the control The chlorophyll content of HAL1 transgenic plants was higher than that of 400 mol·L -1 NaCl solution after 600 mol·L -1 NaCl solution stress. 【Conclusion】 HAL1 was successfully cloned from Saccharomyces cerevisiae; yeast HAL1 played an important role in improving salt tolerance of cotton.