荒漠植物花花柴HTR基因家族表达模式及其增强细菌抗逆性分析

Expression pattern of HTR gene family in desert plant Karelinia caspia and its enhancement of bacterial stress resistance

【目的】荒漠植物花花柴具有耐盐碱、耐高温等优异的广谱抗逆性,是新疆珍贵的天然抗逆植物种质资源。挖掘花花柴耐极端温度和耐盐基因,系统分析其在高温、低温和高盐胁迫下表达模式以及抗逆性,对于利用该基因增强作物抗逆性、稳产性、丰产性,促进荒漠植物基因资源的发掘利用都具有重要意义。【方法】采用盆栽花花柴幼苗进行不同时长的高温(45℃)、低温(4℃)和高盐(400 mmol/L Na^(+))胁迫处理,采集幼苗的根、茎、叶组织,利用RT-PCR等技术克隆花花柴HTR基因家族(KcHTRs),分析KcHTRs基因表达情况,同时通过原核表达体系分析其抗逆性以及表达蛋白质的最佳诱导条件。【结果】(1)从荒漠植物花花柴高温转录组数据中筛选并克隆了KcHTR基因家族的7个成员,通过构建原核表达载体并诱导表达,明确其cDNA长度在708~789 bp之间,所表达的蛋白质分子量在27~29 kD之间;(2)发现在大肠杆菌BL21最适生长温度37℃下该家族蛋白的最佳诱导表达体系:OD600为0.8,IPTG诱导浓度为0.5 mmol/L,诱导时间为8~10 h;(3)通过对重组大肠杆菌的模拟高温、低温、盐胁迫处理,结果显示KcHTRs能够在一定程度上增强宿主对高温、低温和高盐的耐受性。【结论】KcHTRs具有优异的广谱抗逆性,主要具有耐极端温度的抗逆性,且能够积极响应花花柴植株应对逆境胁迫。

[Objective]The desert plant Karelinia caspia has a broad-spectrum of stress resistance,including salt/alkali tolerance and high temperature tolerance,which is a precious natural plant germplasm native to Xinjiang.It has been noted that mining the genes involved in extreme temperature and salt tolerance of K.caspia as well as analyzing their expression under stress is critical for using these genes to enhance crop stress resistance,increase yield,and promote the exploration and utilization of the genetic resources of the desert plants.[Methods]Potted K.caspia seedlings were subjected to different durations of high temperature(45℃),low temperature(4℃),and high salt(400 mmol/L Na^(+))stress.Root,stem,and leaf tissues were collected to analyze the expression of the HTR gene family(KcHTRs)by using RT-PCR and other techniques,and analyze their stress resistance and optimal induction conditions for protein expression by a prokaryotic expression system.[Results](1)Seven of the KcHTR gene family members were screened and cloned from the transcriptomic data obtained from high temperature treatments of K.caspia.By constructing prokaryotic expression vectors and inducing expression,cDNA lengths of the KCHTR gene family were determined to be between 708 and 789 bp,and the molecular weights of the expressed protein was between 27 and 29 kD.(2)It was found that the optimal expression system of E.coli BL21 was 0.8 at OD600,IPTG concentration 0.5 mmol/L,and induction time 8-10 h at the optimum temperature of 37℃.(3)Recombinant E.coli was treated with simulated high temperature,low temperature and salt stress,and the results showed that KcHTRs enhanced the host tolerance to the stresses to certain extent.[Conclusion]The results showed that KcHTRs has broad-spectrum of stress resistance,mainly extreme temperature,and actively responds to stress conditions.