棉花多酚氧化酶基因GhPPO1的克隆及在棉铃虫取食诱导反应中的作用

Cloning of a Polyphenol Oxidase Gene(GhPPO1) of Gossypium hirsutum and Its Role in Cotton after Helicoverpa armigera Feeding

【目的】克隆棉花多酚氧化酶基因(GhPPO1)全长cDNA序列,分析其序列特征,并研究其及多酚氧化酶系对棉铃虫(Helicoverpa armigera)的取食诱导反应,明确其在棉花防御棉铃虫取食中的作用。【方法】根据笔者研究组前期从棉花SSH文库中获得的对昆虫取食诱导响应明显的棉花PPO基因序列片段,设计5′RACE特异引物,进行5′端RACE反应,测序后进行序列拼接。拼接序列在NCBI棉花dbEST数据库中,用Blastn程序进行同源检索,获得一条EST(GenBank登录号:DR461072.1)与测序结果有410 bp重复。用DNAstar进行组装、电子延伸,获得一条棉花PPO基因序列,命名为GhPPO1。在GhPPO1序列两端设计引物进行全长验证,同时设计引物,以棉花基因组DNA为模板,进行PCR扩增测序,验证GhPPO1是否含有内含子。采用BLASTX在NCBI数据库中对验证后的GhPPO1序列进行同源序列分析;ClustalW软件进行多重序列比对;MEGA 4.0软件构建系统进化树;同时对推测的编码区氨基酸序列进行功能位点及理化性质的预测与分析,所用软件包括ANTHEPRO5.0、ExPASy、InterProscan等。利用实时荧光定量PCR方法测定机械损伤、棉铃虫取食和口腔分泌物处理后,棉花叶片中GhPPO1的mRNA表达量;并通过分光光度法测定棉花叶片中PPO酶系的活性变化趋势。【结果】GhPPO1的cDNA序列全长为2 022 bp,推测该基因编码区(ORF)为1 797 bp,编码598个氨基酸,5′UTR含102 bp,3′UTR含123 bp,预测其等电点为6.11,蛋白分子量约67.18 kD,无内含子。GhPPO1编码区氨基酸序列包含CuA和CuB结合位点、3个N-糖基化位点、8个N-豆蔻酰化位点、6个蛋白激酶C磷酸化位点、8个酪蛋白激酶Ⅱ磷酸化位点、1个依赖于cAMP和cGMP的蛋白激酶磷酸化位点及1个酰胺化位点。GhPPO1的CuA和CuB离子结合区,有PPO蛋白关键氨基酸组氨酸和半胱氨酸。GhPPO1蛋白与其他植物的PPO蛋白序列最大相似度几乎均在50%以上,与其他植物PPO蛋白进化关系相对较远。机械损伤棉花叶片后,GhPPO1表达量呈现出先升高后下降的趋势,至6 h表达量最高,为无处理对照的3.29倍;1龄末期棉铃虫幼虫取食棉花后,叶片中该基因的表达量呈现先升高后下降,然后又升高的趋势,至12 h表达量最高,为无处理对照的6.04倍;棉铃虫取食3 h和6 h后的表达量低于机械损伤处理后相同时间的表达量。棉铃虫取食和机械损伤棉花叶片后,PPO酶活性均呈现出升高趋势,且机械损伤处理酶活性在各时间段均高于棉铃虫幼虫取食处理。与未做任何处理的正常叶片相比,机械损伤和清水共同处理的叶片GhPPO1表达量及PPO酶活性显著升高,而机械损伤和棉铃虫幼虫口腔分泌物共同处理的叶片GhPPO1表达量及PPO酶活性无显著变化,表明口腔分泌物对GhPPO1表达量及PPO酶活性有抑制作用。【结论】获得了GhPPO1的全长cDNA序列,证明GhPPO1是棉花防御棉铃虫取食的关键PPO基因,推测棉铃虫口腔分泌物中存在抑制棉花PPO酶活性升高物质,该物质在棉铃虫适应寄主植物产生的防御反应中发挥作用。

【Objective】 The objective of this study is to clone and characterize GhPPO1 from cotton（Gossypium hirsutum）, then to study the dynamic changes of GhPPO1 mRNA expression level and PPO activity induced by Helicoverpa armigera in order to clarify the function of this gene in defensing response in cotton. 【Method】 A fragment of PPO gene which has an obvious response to insect feeding from cotton SSH library was got. Specific primers were designed to conduct the 5′RACE reaction. After sequencing and assembly, homologous retrieval was carried out in the NCBI cotton dbEST database and an EST（GenBank accession number： DR461072.1） which have a 410 bp overlap with targeted PPO gene were found. After assembly and electronic extension by DNAstar software, the complete sequences of cotton PPO gene, named GhPPO1, was got. To verify the sequence authenticity of GhPPO1, primers were designed at the two ends of the gene. Using genomic DNA as template, PCR was done to check whether there exist introns in GhPPO1. After verification, the tool of BLASTX was used to analyze the sequence homologous and ClustalW software was used to do multiple sequence alignment and MEGA 4.0 was used to construct a phylogenetic tree and other softwares including ANTHEPRO5.0, ExPASy and InterProscan were used to predict the existence of functional sites and physicochemical properties in the speculated coding regions. The GhPPO1 mRNA expression level and PPO activity in cotton leaves were detected by real-time quantitative PCR and spectrophotometric method, respectively. 【Result】 The complete cDNA sequence of GhPPO1 is 2 022 bp, with a 1 797 bp open reading frame which encodes 598 amino acids（M= 67.18 kD and pI= 6.11）. The 5′ and 3′ untranslated regions were 102 and 123 bp, respectively. There were no introns in this gene. In the amino acid coding sequence, there were CuA and CuB binding sites, 3 N-glycosylation sites, 8 N-nutmeg acylation sites, 6 protein kinase C phosphorylation sites, 8 casein kinase Ⅱ phosphorylation sites, 1 cAMP and cGMP dependent protein kinase phosphorylation site and 1 amidation site. In the CuA and CuB binding regions, there existed the histidine and cysteine which play a key role in PPO protein. The similarity of PPO protein between cotton and other plants were more than 50%, however, the relationship of GhPPO1 protein was relatively far away with other kinds of PPO proteins. After mechanical damage, cotton leaf GhPPO1 expression showed a trend of rise and fall. The expression level was the highest at 6 h and the maximum fold change was 3.29 times compared with un-treated leaves. GhPPO1 mRNA level showed a variable trend which was increased firstly and then decreased and then increased at last after 1st late instar cotton bollworm larvae feeding. Twelve hours after feeding, the expression reached the highest level which was 6.04 times higher than the un-treated leaves. However, the mRNA expression level was lower in cotton bollworm feeding leaves than in mechanically injured ones at both 3 h and 6 h. PPO activity showed an increasing trend after both insect feeding and mechanical wounding treatment. The enzyme activity was lower in former treatment. Compared with the untreated cotton leaves, the mRNA expression level and enzyme activity significantly up-regulated after mechanical injury and water jointly treated leaves. However, there were no significant difference between the oral secretion treated and untreated cotton leaves in both GhPPO1 mRNA expression level and PPO activity. It indicated that oral secretion from cotton bollworm larvae might have a suppression role in both GhPPO1 mRNA expression and enzyme activity.【Conclusion】The full-length cDNA sequence of GhPPO1 which plays an important role in defensing cotton bollworm was cloned from cotton. It is speculated that some inhibitors may exist in the oral secretion of cotton bollworm larvae, which may cope with the defense system induced by insects.