中国农业科学 ›› 2016, Vol. 49 ›› Issue (14): 2651-2661.doi: 10.3864/j.issn.0578-1752.2016.14.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

酵母耐盐相关基因HAL1在棉花中的功能表达

穆 敏,舒 娜,王 帅,郭丽雪,樊伟丽,阴祖军,王俊娟,王德龙,叶武威   

  1. 中国农业科学院棉花研究所/棉花生物学国家重点实验室/农业部棉花遗传改良重点开放实验室,河南安阳 455000
  • 收稿日期:2016-02-15 出版日期:2016-07-16 发布日期:2016-07-16
  • 通讯作者: 叶武威,Tel:0372-2562283;E-mail:yew158@163.com
  • 作者简介:穆敏,Tel:15890348192;E-mail:15890348192@163.com
  • 基金资助:
    国家转基因生物新品种培育科技重大专项(2014ZX0800504B)

The Function Expression of Salt-Tolerant Yeast Gene Halotolerance ( HAL1 ) in Cotton

MU Min, SHU Na, WANG Shuai, GUO Li-xue, FAN Wei-li, YIN Zu-jun, WANG Jun-juan, WANG De-long, YE Wu-wei   

  1. Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology/Key Laboratory for Cotton Genetic Improvement, Ministry of Agriculture, Anyang 455000, Henan
  • Received:2016-02-15 Online:2016-07-16 Published:2016-07-16

摘要: 【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pBI121-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T0棉花转基因种子。用100 mmol·L-1 NaCl盐溶液对转基因T0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 mmol·L-1 NaCl盐溶液对转基因棉花T0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。根据基因核苷酸序列设计2对引物对T0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 mmol·L-1 NaCl和400 mmol·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 mmol·L-1 NaCl溶液胁迫后,转HAL1植株叶绿素含量反而高于400 mmol·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。

关键词: 酵母, HAL1, 陆地棉, 花粉瞬时表达, 分子检测

Abstract: 【Objective】 The objective of this study is to clone Saccharomyces cerevisiae halotolerance (ScHAL1) gene and transformed into cotton , explore the function of the gene in cotton, to further explore function of the salt-tolerant yeast genes in higher plants. 【Method】According to total length of mRNA sequence information of ScHAL1 in NCBI, the gene was cloned using RT-PCR technology from Saccharomyces cerevisiae As2.375, double enzyme digestion was made with XbaⅠand SmaⅠfor pBI121::GFP, and pBI121-ScHAL1::GFP fusion expression vector was constructed by In-Fusion technique. With weak auto-fluorescence upland cotton varieties, Y-2067, ZA-23 and GZ-2 pollen as materials, using the gene bombarding technique to study transient expression of cotton pollen. The expression vector pBI121-ScHAL1::GFP was transformed into cotton salt-sensitive material Zhong s9612 with gene gun in vivo conversion technology, T0 generation cotton genetically seeds were modified. Solution with 100 mmol·L-1 NaCl was used to test the salt resistance of seeds in the transgenic T0 generation seed germination experiment, molecular detection was carried out, and semal salt resistance of transgenic plants was analysis. 【Result】 ScHAL1 cloned from Saccharomyces cerevisiae As2.375 and ScHAL1 is 885 bp in length, which encoding 294 amino acids. After its sequence analysis, it was found that the largest proportion of the whole HAL1 protein is serine, and it is alkaline and positively charged and it is a hydrophilic protein. According to the results of protein secondary structure prediction, it is speculated that the structure of the protein function domain may mainly made up of random curl and beta sheet. Cotton pollen instantaneous expression results showed that after conversion of HAL1 gene, three land cotton powder green fluorescence were obviously enhanced, suggesting that the gene expressed in these three upland cotton pollen. With 100 mmol·L-1 NaCl, transgenic T0 generation seed germination ability obviously stronger than receptor s9612 selfing seed material, which preliminary showed that HAL1 could also improve the seed salt resistance. According to the gene nucleotide sequences, two pairs of primers were designed for molecular detection of T0 generation. Direct sequencing of PCR product was conducted and the sequencing results preliminarily evidence that the transgene is successful. With the semal salt resistance analysis, it was found that the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl and 400 mmol·L-1 NaCl were higher than the control, and the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl were higher than that in 400 mmol·L-1 NaCl. 【Conclusion】HAL1 gene was Successfully cloned from Saccharomyces cerevisiae, yeast HAL1 gene plays an important role in improving cotton salt resistance.

Key words: yeast, HAL1, upland cotton, pollen instantaneous expression, molecular detection