摘要
棉花是世界上重要的经济作物之一,然而棉花的产量和品质经常受到盐害胁迫的影响,利用基因工程技术研发耐盐棉花品种,是解决这个问题的有效方法。ST6-40基因是通过一种功能基因挖掘方法从小盐芥中分离得到,该方法基于构建大量独立株系组成的转基因种子文库,通过高通量遗传筛选法筛选耐盐株系,得到的基因与耐盐功能直接相关。将携带有ST6-40基因的pCB2004-ST6-40重组载体通过农杆菌介导法转入陆地棉品种R15,获得转基因棉花再生株系;采用棉花生理指标测定法和直接耐盐鉴定方法对获得的转基因棉花株系进行耐盐性鉴定,转基因棉花株系T1和T2通过测定叶片游离脯氨酸含量进行鉴定,转基因棉花株系T_(3)采用盐水浇灌直接鉴定,依据盐害级数计算盐害指数进行耐盐性判定。结果表明,共进行了350个转化事件,转化率为19%,获得65个阳性T_(0)株系,其中再生植株中90%为阳性苗;经T_(1)、T_(2)、T_(3)连续3代基因组水平PCR检测及RNA水平上表达量鉴定,表明目的基因能够稳定遗传。转基因阳性植株经T_(1)、T_(2)、T_(3)耐盐性检测,获得6个耐盐性较受体对照R15显著增强的转基因纯合株系,4个转基因株系耐盐性优于耐盐对照品种中99807,表明转入ST6-40基因能有效提高棉花品种的耐盐性。
Cotton is one of the important economic crops in the world,but the yield and quality of cotton are often affected by salt stress.Developing salt-resistant cotton varieties by genetic engineering technology is an effective method to solve this problem.ST6-40 gene was isolated from Thellungiella halophila by a functional gene mining method based on the construction of transgenic seed libraries composed of numerous independent lines and the screening of salt-tolerant lines by high-throughput genetic screening,and the obtained objective genes were directly associated with salt-tolerant function.In this study,the pCB2004-ST6-40 recombinant vector was transformed into an upland cotton variety R15 by Agrobacterium-mediated method,and the regenerated lines of transgenic cotton were obtained.To identify the salt tolerance of the transgenic cotton lines,physiological index assay and direct salt tolerance identification were taken.T_(1) and T_(2) generations of transgenic cotton lines were identified by measuring free proline content of leaves,while T_(3) generation of transgenic cotton lines were directly identified by saline irrigation,and the salt tolerance was determined by calculating the salt damage index according to the salt damage series.The results showed that 350 transformation events were performed,and the transformation rate was 19%,65 positive T_(0) generation lines were obtained,and 90%of the regenerated plants were positive plants.PCR detection ofT_(1)、T_(2) and T_(3) for three consecutive generations at the genome level and expression identification at the RNA level showed that the target gene could be inherited stably.Through the salt-tolerance detection of T_(1)、T_(2) and T_(3)generation of transgenic positive plants,six transgenic homozygous lines with significantly enhanced salt tolerance compared with the receptor cotton R15 were obtained,and the salt tolerance of four transgenic lines was better than that of the salt tolerant control variety 99807,indicating that transgenic ST6-40 gene could effectively improve salt tolerance of cotton varieties.
作者
朱永红
吴慎杰
李静
张换样
焦改丽
竹梦婕
秦丽霞
ZHU Yonghong;WU Shenjie;LI Jing;ZHANG Huanyang;JIAO Gaili;ZHU Mengjie;QIN Lixia(Institute of Cotton,Shanxi Agricultural University,Yuncheng 044000,China;State Key Laboratory of Cotton Biology,Anyang 455000,China)
出处
《山西农业科学》
2022年第12期1608-1613,共6页
Journal of Shanxi Agricultural Sciences
基金
国家自然科学基金项目(31601350)
国家转基因生物新品种培育重大专项(2016ZX08005)
第六批山西省青年拔尖人才项目(20180612)
山西省农业科学院博士基金项目(YBSJJ1804)
山西省高等教育创新项目(2021L115)
山西省基础研究计划项目(20210302123381)
棉花生物学国家重点实验室开放课题基金(CB2022A28)。
关键词
棉花
ST6-40
过量表达
转基因植株
耐盐性
cotton(Gossypium hirsutum L.)
ST6-40
overexpression
transgenic plants
salt tolerance
