转α-微管蛋白基因SoTUA甘蔗T2代的生理生化特性分析

Analysis of Physio-biochemical Characteristics of T2α-tubulin SoTUA Transgenic Sugarcane

本研究对已获得的转α-微管蛋白基因SoTUA的T2代甘蔗株系进行PCR扩增及测序,从检测阳性的转基因甘蔗中选6个转基因株系(T1、T2、T3、T4、T5、T6)与非转基因野生型甘蔗对照(WT)进行了低温胁迫(4℃)处理,在处理0、5、10 d测定甘蔗叶片丙二醛(MDA)和渗透调节物(可溶性糖、可溶性蛋白)含量以及过氧化物酶(POD)和超氧化物歧化酶(SOD)活性,并采用RT-qPCR法分析了低温处理下甘蔗叶片α-微管蛋白基因的表达情况。结果表明:在4℃低温处理下,6个转SoTUA基因甘蔗与WT中α-微管蛋白的转录因子表达量随着胁迫时间的延长上调表达。胁迫后5、10 d转基因株系的相对表达量始终高于WT甘蔗,低温处理10 d时转基因T1显著高于其他株系(P<0.05)。在低温胁迫下,甘蔗的MDA含量逐渐增高,而转基因甘蔗增幅显著低于WT甘蔗,10 d时转基因株系MDA的含量比WT低41.77%(P<0.05);POD活性在不同株系之间变化趋势不同,但转基因甘蔗POD活性显著高于WT甘蔗(P<0.05);转基因T1、T2、T4、T6株系的SOD活性随着胁迫天数增加呈现先增加后减少的趋势,而WT与转基因T3、T5的SOD活性逐渐升高;甘蔗的可溶性糖含量逐渐上升,转基因甘蔗的可溶性蛋白含量在0 d显著高于野生型甘蔗,胁迫后变化趋势在株系之间有所差异;转基因甘蔗SoTUA相对表达量与可溶性糖含量呈极显著的正相关。利用隶属函数综合分析评价,抗寒性排序结果为:T3>T2>T1>T5>T6>T4>WT。在低温胁迫下,转基因甘蔗SoTUA基因的上调表达能提高甘蔗的抗寒性,且具有遗传稳定性。

Six SoTUA transgenic lines(T1,T2,T3,T4,T5,T6)were selected from the positive SoTUA transgenic sugarcane lines of T2 generation after PCR amplification and sequencing.The transgenic lines and control(wild type,WT)were treated with low temperature stress(4℃),and the level of malondialdehyde(MDA)and osmotic adjustment substances(soluble sugar,soluble protein)and activities of peroxidase(POD)and superoxide dismutase SOD were determined,and RT-qPCR analysis was done for SoTUA gene expression in sugarcane leaves at 0,5 and 10 days of the treatment.The relative expression ofα-tubulin transcription factor in the SoTUA transgenic sugarcane and WT was up-regulated,getting higher with the cold stress time under low temperature treatment at 4℃.The relative expression level was always higher in the SoTUA transgenic lines than that in WT at 5 and 10 days of the treatment.At the 10 th day,the relative expression level of the transgenic line T1 was significantly higher than that of other transgenic strains(P<0.05).The content of MDA increased gradually under cold stress,but it was found significantly lower in the transgenic sugarcane than that in WT by 41.77%(P<0.05).The trend of POD activity varied among different lines,but it was higher in the SoTUA transgenic lines than in WT(P<0.05).The SOD activity of transgenic lines T1,T2,T4 and T6 increased first and then decreased while SOD activity of WT and transgenic lines T3 and T5 increased gradually.The soluble sugar content increased gradually and the trend of soluble protein varied in different strains.The expression of SoTUA gene was positively correlated with soluble sugar.Using the membership function comprehensive analysis method,the ranking result for cold resistance was obtained as T3>T2>T1>T5>T6>T4>WT.Up-regulated expression of SoTUA gene in the transgenic sugarcane could improve cold resistance,and the genetic stability is good.