菘蓝IiLEA基因的克隆及胁迫表达分析

Cloning and Expression Analysis under Stress of IiLEA Gene in Isatis indigotica

胚胎发育晚期丰富蛋白(LEA蛋白),是指胚胎发育后期种子中大量积累的一系列蛋白质,大量研究表明这些蛋白质的积累与渗透胁迫耐受性密切相关.该实验以250 mmol/L NaCl溶液处理9 h的菘蓝幼苗为材料,经RT-PCR方法扩增得到LEA基因,命名为IiLEA.经测序确认该基因含有648 bp的开放阅读框,编码由215个氨基酸组成的亲水性蛋白质.对培养30 d的菘蓝无菌幼苗进行自然干旱处理和盐胁迫处理,Northern杂交结果显示,正常生长条件下,IiLEA基因在菘蓝幼苗体内不表达,随着胁迫时间的延长其表达量逐渐增加,到9 h时表达量达到峰值,干旱处理和盐处理有相似结果,表明该基因可能受逆境胁迫诱导表达.

Later embryogenesis abundant proteins（LEA proteins） accumulate to high concentrations in plant embryo during the later stage of seed development.Most of them accumulate under osmotic stress treatment.In this study,we amplified LEA gene by RT-PCR,named as IiLEA from the seedlings of Isatis indigotica Fort.,which were treated with 250 mmol/L NaCl for 9 hours.The gene,including a 648 bp opening reading frame,encodes a hydrophilic protein containing 215 amino acid.The seedlings of I.indigotica Fort.were treated with natural drought and salt stress after cultivated in aseptic environment for 30 days.Northern hybridization analysis showed that IiLEA gene did not express in the normal growth conditions in Isatis seedlings,but its expression level gradually increased with stress time and reached peak after 9 h treatment.There were similar results in drought and salt treatments.It indicated that the expression of the gene may be induced by environmental stress.