蒺藜苜蓿MtSAG113基因的转化及表达特征分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0481

摘要/Abstract

摘要：

Senescence Associated Gene 113（SAG113）基因属于PP2Cc超家族,该基因的研究主要集中在植物衰老领域。为分析蒺藜苜蓿（Medicago truncatula）MtSAG113基因的表达特征,探究MtSAG113基因的功能。该基因从蒺藜苜蓿中克隆得到,以烟草（Nicotiana tabacum L.）和蒺藜苜蓿R108为实验材料,通过农杆菌介导的叶盘法转化烟草获得具有抗性的转基因植株,并对逆境胁迫和外源激素诱导下蒺藜苜蓿MtSAG113基因的表达情况进行检测。结果显示：PCR检测成功获得5株转基因植株并在在烟草中成功表达,且转基因植株相比于野生型出现矮小和叶片早衰的表型;MtSAG113基因在衰老叶片中表达水平远远高于未衰老叶片;未处理的叶片中,该基因的表达水平随着时间的延长逐渐提高;干旱胁迫下,该基因表达水平随着时间推移显著提高;高盐胁迫下,该基因表达水平明显上调,在12 h达到峰值;该基因对6-BA诱导尤为敏感,表达水平随着时间推移显著提高,在4 h达到峰值,约为未处理的1 000倍;而在ABA和IAA诱导条件下,MtSAG113呈现类似的表达特征,从4 h开始提高,12 h达到峰值;亚细胞定位结果显示,该基因位于细胞核内。以上结果表明：MtSAG113基因可能在蒺藜苜蓿衰老调控上发挥着一定的作用;而高盐、干旱和外源激素对MtSAG113基因表达具有一定的调节作用,以上研究为进一步分析MtSAG113基因的功能提供基础。

关键词: MtSAG113基因, 蒺藜苜蓿, 烟草, 逆境胁迫, 外源激素

Abstract:

Senescence Associated Gene 113（SAG113）genes belong to PP2Cc super family and they are involved in plant senescence. This work aims to analyze the expression characteristics of the gene MtSAG113 of Medicago truncatula and to explore the function of the MtSAG113. This gene was cloned from M. truncatula. Using Nicotiana tabacum L. and M. truncatula R108 as experimental materials,the Agrobacterium-mediated leaf disc method was used to transform tobacco to obtain transgenic plants with resistance. The expression of MtSAG113 from M. truncatula was detected under induction of stress and exogenous hormones. The results showed that 5 transgenic plants successfully expressed in tobacco were obtained by PCR detection and the transgenic plants presented the phenotype of shortness and premature leaf senescence compared with the wild ones. The expressions of MtSAG113 in the senescence leaves were much higher than those in non-senescence leaves. In untreated leaves,the expression of this gene gradually increased with time and the gene was significantly up-regulated expressed under drought stresses. The expressions were significantly up-regulated under salt stresses and reached the peak when suffered for 12 h. The gene was particularly sensitive to 6-BA induction,and the expression increased significantly with time increasing,reached a peak at 4 h,which was about 1 000 times higher than those untreated. Treated with ABA and IAA,MtSAG113 exhibited a similar expression pattern,the expression increased from 4 h,reached a peak at 12 h. The subcellular localization results demonstrated that the gene was located in the nucleus. Above results indicate that MtSAG113 may play a certain role in senescence regulation in M. truncatula,and high salinity,drought and exogenous hormones regulate the expression of MtSAG113,which provides a basis for further function analysis of MtSAG113.

Key words: MtSAG113 gene, Medicago truncatula, tobacco, stress, exogenous hormone

李舒文, 李殷睿智, 董笛, 王梦迪, 晁跃辉, 韩烈保. 蒺藜苜蓿MtSAG113基因的转化及表达特征分析[J]. 生物技术通报, 2022, 38(1): 108-114.

LI Shu-wen, LI Yin-rui-zhi, DONG Di, WANG Meng-di, CHAO Yue-hui, HAN Lie-bao. Transformation and Expression Pattern Analysis of Gene MtSAG113 from Medicago truncatula[J]. Biotechnology Bulletin, 2022, 38(1): 108-114.

图/表 12

图1 转基因植株PCR检测 M：DNA marker DL 2000 plus II;1：WT;2-8：再生植株

Fig.1 PCR detection of transgenic plants M ：DNA marker DL 2000 plus II; 1：WT; 2-8：regeneration plant

图2 转基因烟草RT-PCR检测 M：DNA marker DL 2000 plusⅡ;1：阴性对照;2-6：转基因烟草

Fig.2 RT-PCR detection of transgenic tobacco M ：DNA marker DL 2000 plus Ⅱ; 1：negative control; 2-6：transgenic tobacco

图3 转基因烟草表型 1：野生型烟草;2-4：转基因烟草

Fig.3 Phenotype of transgenic tobacco 1: Wild type tobacco; 2-4: transgenic tobacco

图4 MtSAG113基因在蒺藜苜蓿叶片中的表达分析

Fig.4 Expression analysis of MtsAG113 gene in Medicago truncatula leaves

图5 蒺藜苜蓿成熟衰老叶片对比 1：成熟叶片：2：衰老叶片

Fig.5 Comparison of mature and senescent leaves of Medicago truncatula 1: Mature leaves; 2: senescent leaves

图6 蒺藜苜蓿MtSAG113基因未处理条件下的表达分析

Fig.6 Expression analysis of MtsAG113 gene in Medicago truncatula under untreated conditions

图7 蒺藜苜蓿MtSAG113基因的干旱胁迫表达

Fig.7 Drought-stress expression of MtsAG113 gene in Medicago truncatula

图8 蒺藜苜蓿MtSAG113基因的盐胁迫表达

Fig.8 Expression of MtsAG113 gene in Medicago truncatula under salt stress

图9 6-BA诱导对蒺藜苜蓿MtSAG113基因表达影响

Fig.9 Effects of 6-BA induction on MtsAG113 gene expres-sion in Medicago truncatula

图10 IAA诱导对蒺藜苜蓿MtSAG113基因表达影响

Fig.10 Effects of IAA induction on MtsAG113 gene expre-ssion in Medicago truncatula

图11 ABA诱导对蒺藜苜蓿MtSAG113基因表达影响

Fig.11 Effects of ABA induction on MtsAG113 gene expre-ssion in Medicago truncatula

图12 亚细胞定位结果

Fig.12 Results of subcellular localization

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