桑树低温诱导基因Wap在不同地理来源桑品种幼叶中的表达差异

为探究不同桑树品种抗寒性差异的分子机制,以4个不同地理来源桑品种的幼苗叶片为材料提取总RNA,利用RT-PCR方法分析正常环境和低温(4℃)胁迫下桑树低温诱导基因Wap(winter accumulating protein)在各品种间的表达差异。正常环境下,来自吉林省的向海桑中Wap的表达水平最高,在来自新疆维吾尔自治区的实生桑和来自江苏省的丰驰桑中该基因也有较高水平的表达,而在来自广东省的杂交桑塘10×伦109中则检测不到该基因的表达。低温胁迫2～6 d,4个桑品种幼苗叶片中Wap的表达均上调,至胁迫8～10 d时Wap的表达达到最高水平;低温胁迫12 d,Wap在广东杂交桑的表达开始降低,低温胁迫16 d,在丰驰桑与新疆实生桑的表达也开始降低,而来自寒冷地域的向海桑在低温胁迫20 d该基因的表达才开始降低;低温胁迫24 d,在广东杂交桑中已检测不到Wap的表达,在丰驰桑与新疆实生桑中仅有微量表达,而在向海桑中仍有较高水平的表达。初步推测Wap在不同桑树品种的表达差异是各品种抗寒性差异的重要因素之一。构建重组质粒pGEX-4T-2-Wap在大肠杆菌中表达了分子质量约27 kD的WAP蛋白,有助于进一步研究其生物学功能。

Expressional Difference of Low-temperature Induced Gene Wap in Young Leaves of Mulberry from Different Geographical Provenances

In order to understand the molecular mechanism of cold tolerance difference among different mulberry varieties,expressional difference of mulberry low-temperature induced gene Wap(winter accumulating protein) under normal environment and low temperature(4 ℃) was analyzed using RT-PCR approach with the total RNA extracted from leaves of young mulberry plants of four different geographical provenances.The results showed that,under normal environment,Wap had the highest expression level in Xianghai Sang from Jilin Province,with a relatively higher expression level in seedling mulberry from Xinjiang Uygur Autonomous Region and in Fengchi Sang from Jiangsu Province.Wap could not be detected in the hybrid mulberry variety Tang 10×Lun 109 from Guangdong Province.On the 2nd to 6th day after low-temperature treatment,the expression levels of Wap in all 4 varieties were up-regulated.They reached the maximum levels on the 8th to 10th day.After low-temperature treatment for 12 d,the expression level of Wap in hybrid mulberry from Guangdong Province began to decline,while the decline happened in Fengchi Sang and seedling mulberry from Xinjiang Uygur Autonomous Region on the 16th day.The expression level of Wap in Xianghai Sang from cold region began to decline on the 20th day.On the 24th day after low-temperature treatment,there was no expression in hybrid mulberry from Guangdong Province,and only trace expression could be detected in Fengchi Sang and Xinjiang seedling mulberry,while high expression level of Wap was still maintained in Xianghai Sang.These results suggest that the differential expression of Wap in different mulberry varieties is one of the important reasons leading to difference in cold tolerance.A recombinant plasmid pGEX-4T-2-Wap was constructed and WAP protein of molecular weight of about 27 kD was obtained from expression in E.coli,being helpful for further studies of its biological functions.