沙冬青脱水素基因转化紫花苜蓿的耐寒性研究

Study on Cold-stress Tolerance of Transgenic Alfalfa(Medicago sativa L.) with AmDHN Gene

以紫花苜蓿品种‘中苜2号’为野生型材料,采用农杆菌介导法将沙冬青脱水素基因(dehydrin,AmDHN)导入紫花苜蓿基因组中并获得转基因植株,通过PCR和Southern blot杂交鉴定转基因植株,利用RT-PCR和qRT-PCR检测转基因植株中AmDHN基因及低温胁迫相关基因的表达量,并测定低温胁迫下苜蓿叶片的脯氨酸(Pro)和丙二醛(MDA)含量,从分子水平和生理指标两个层面研究转基因植株的抗寒特性,为进一步获得抗寒性较强的转基因苜蓿新材料提供依据。结果显示:(1)AmDHN基因已整合在转基因苜蓿植株基因组中,而且在不同的转基因株系中AmDHN的表达量也各不相同。(2)低温(4℃)处理后转基因植株中冷胁迫相关基因CBF2、CBF3、ProDH和CAS17的表达量明显高于同期野生对照;CBF2、CBF3和CAS17表达量在冷处理5h后都显著增加并达到最大值,而ProDH表达量在冷处理7d时最高,它们的最高值分别是对照的2.5、4、1.6和3倍左右。(3)苜蓿叶片的Pro和MDA含量均随低温处理时间延长而逐渐增加,转AmDHN基因苜蓿叶片的Pro含量始终高于同期野生型植株,而其MDA含量却始终低于同期野生型植株,且两类植株间差异均在胁迫14d时达到显著水平。因此,推测转AmDHN基因苜蓿中积累的AmDHN蛋白可能对一些酶的活性及膜系统起冷冻保护作用,从而使得转AmDHN基因紫花苜蓿的植株抗寒性提高,同时AmDHN也可能通过调控与低温相关基因的表达间接调节植物的耐低温能力。

Alfalfa varieties ‘Zhongmu No.2’ was acted as the wild-type material.Agrobacterium-mediated method was used to introduce dehydrin gene（AmDHN） into alfalfa.PCR and southern blot methods were used to detect the presence of AmDHN in transgenic alfalfa plants.The expressions of AmDHN and coldrelated genes were detected in transgenic plants of T0 generations through RT-PCR and qRT-PCR.The content of proline and MDA of two plants were detected.Cold resistance of the transgenic alfalfa plants was detected by molecular level and physiological characteristics,which will provide a basis for new transgenic alfalfa material with resistance to cold stress.The main results are summarized as follows：（1）Expression of AmDHN was determined at different levels in transgenic alfalfa plants.（2）After being treated with 4 ℃,expressions of cold-related genes（CBF2,CBF3,ProDH and CAS17） were dramatically higher than WT.Expressions of CBF2,CBF3 and CAS17 increased significantly after being treated 5 h,reached an extremely significant level（P＜0.05）,as compared with WT plants.The level of ProDH transcripts reached the maximum at 7 d after treatment by 4 ℃,their maximum were 2.5,4,1.6 and 3 times than WT,respectively.（3）The contents of both proline and MDA were increasing with extending treatment time.The content of proline of transgenic plants was significantly higher than that of WT.The content of MDA was significantly lower than that of WT,both goes up to an extremely significant level at 14 d after the treatment.These results suggested that transgenic plants may be more resistance to cold stress compared with WT.These imply that AmDHN proteins may protect the enzyme activity and membrane systems from freezing injury.Simultaneously,AmDHN may regulate indirectly cold tolerance of the plants through regulating the expressions of cold-related genes.