MsRCI2A/B/C基因超量表达对紫花苜蓿耐旱性的影响

Effect of overexpression of MsRCI2A/B/C genes on drought tolerance of alfalfa(Medicago sativa L.)

【目的】分析紫花苜蓿MsRCI2A、MsRCI2B、MsRCI2C的表达差异并研究其抗旱功能,为苜蓿抗旱机制解析及分子育种奠定基础。【方法】采用qPCR的方法,分析紫花苜蓿MsRCI2A、MsRCI2B、MsRCI2C基因在20%PEG6000溶液模拟干旱条件下表达的差异,采用农杆菌介导法获得MsRCI2A、MsRCI2B和MsRCI2C基因超量表达的转基因苜蓿A12、A22、B13、B19、C2和C10,观察正常生长条件(干旱胁迫0 d)下和20%PEG6000干旱胁迫6,12 d的野生型株系WT(对照)及超量表达的转基因紫花苜蓿的表型,测定超表达苜蓿和WT的叶绿素含量、相对电导率、丙二醛(MDA)含量、抗氧化酶(超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT))活性及渗透调节系统(可溶性糖和脯氨酸含量)在干旱胁迫条件下的变化。【结果】在20%PEG6000模拟干旱条件下,MsRCI2A/B/C基因在紫花苜蓿叶和根中表达变化趋势相反,但3个基因在同一组织中的表达趋势相似。紫花苜蓿叶片中的MsRCI2A/B/C基因在干旱胁迫处理后均明显上调,其中MsRCI2A/C基因在干旱胁迫处理2 h时即极显著上升(P<0.01),而MsRCI2B的表达相对缓慢,在处理8 h时极显著上升(P<0.01)。在根中,MsRCI2A/B/C基因在干旱胁迫处理2 h时相对表达量极显著下降(P<0.01)。在正常生长条件(干旱胁迫0 d)下,6个转基因苜蓿叶绿素含量、相对电导率和MDA含量、可溶性糖含量和脯氨酸含量与野生型苜蓿(WT)相比无明显差异,但转基因苜蓿SOD、POD、CAT活性明显高于WT(P<0.05),其中转MsRCI2B/C基因苜蓿叶片中的SOD活性及叶和根中POD活性是WT的1.97~2.27倍。转基因株系A12、A22、B13、B19、C2和C10与野生型苜蓿(WT)同时进行20%PEG6000模拟干旱胁迫处理12 d后,各转基因株系的叶绿素含量极显著高于WT(P<0.01),为WT的3.00~3.46倍,而相对电导率和丙二醛含量则极显著下降了73%和55%。转基因苜蓿叶和根中的可溶性糖和脯氨酸含量及SOD、POD和CAT活性均显著升高(P<0.05),其中MsRCI2A/C转基因苜蓿叶和根中的CAT活性是WT的2~3倍,MsRCI2B/C转基因苜蓿叶和根中的脯氨酸含量是WT的3倍。【结论】MsRCI2A、MsRCI2B和MsRCI2C的过表达可以增强苜蓿的耐旱性。

【Objective】This study analyzed the expression differences of MsRCI2A,MsRCI2B and MsRCI2C in alfalfa and studied its drought resistance function to lay foundation for analyzing drought resistant mechanism of alfalfa and molecular breeding.【Method】The qPCR method was used to analyze expression differences of alfalfa MsRCI2A,MsRCI2B and MsRCI2C genes under simulated drought conditions with 20%PEG6000 solution.Agrobacterium-mediated method was used to obtain transgenic alfalfa A12,A22,B13,B19,C2 and C10 with overexpression of MsRCI2A,MsRCI2B and MsRCI2C genes.The phenotypes of WT and overexpressed transgenic alfalfa under normal growth conditions(drought stress 0 d)and 20%PEG6000 drought stress for 6 and 12 d were observed,and the changes in chlorophyll content,relative conductivity,malondialdehyde content,activities of antioxidant enzymes(SOD,POD and CAT)and osmotic adjustment system(soluble sugar and proline contents)of overexpressing alfalfa and wild-type strain WT(control)were determined and analyzed.【Result】Under 20%PEG6000 simulated drought conditions,changes in expression of MsRCI2A/B/C genes in leaves and roots were opposite,but the expression trends in same tissues were similar.The MsRCI2A/B/C genes in leaves of alfalfa were significantly up-regulated by drought stress,and MsRCI2A/C genes increased significantly 2 h after drought stress(P<0.01).However,the expression of MsRCI2B was relatively low and it increased significantly 8 h after treatment(P<0.01).In roots,the expression of MsRCI2A/B/C genes decreased significantly 2 h after drought stress(P<0.01).Under normal growth conditions(drought stress 0 d),relative conductivity and contents of chlorophyll,malondialdehyde,soluble sugar and proline of six transgenic alfalfa were not significantly different from wild type alfalfa(WT).However,transgenic plants had higher SOD,POD and CAT activities than WT(P<0.05),among which SOD activity in leaves of transgenic MsRCI2B/C alfalfa and POD activity in leaves and roots were 1.97-2.27 times of that of WT.Transgenic lines A12,A22,B13,B19,C2 and C10 and wild-type alfalfa(WT)were simultaneously treated with 20%PEG6000 to simulate drought stress for 12 days,and chlorophyll contents of transgenic lines were significantly higher than that of WT(P<0.01),which were 3.00-3.46 times of that of WT.The relative conductivity and malondialdehyde content decreased significantly by 73%and 55%.The contents of soluble sugar and proline and activities of SOD,POD and CAT in leaves and roots of transgenic alfalfa were significantly increased(P<0.05).CAT activity in leaves and roots of transgenic MsRCI2A/C alfalfa were 2-3 times of that of WT,and the proline content in leaves and roots of transgenic MsRCI2B/C alfalfa were 3 times of that of WT.【Conclusion】Overexpression of MsRCI2A,MsRCI2B,and MsRCI2C enhanced drought tolerance of alfalfa.