基于转录组分析的低铁胁迫下Bt棉苗杀虫蛋白表达量下降机制  

Mechanism of reduced insecticidal protein expression in Bt cotton under low-iron stress based on transcriptome analysis

作　　者：王永慧[1] 贺江 张向向 娄向弟 高进[1] 孙艳茹[1] 曹婷[1] 施洋 WANG Yong-Hui;HE Jiang;ZHANG Xiang-Xiang;LOU Xiang-Di;GAO Jing;SUN Yan-Ru;CAO Ting;SHI Yang(Institute of Agricultural Sciences of Jiangsu Coastal Area/Observation and Experimental Station of Saline land of Costal Area,Ministry of Agricul-ture and Rural Affairs,Yancheng 224001,Jiangsu,China)

机构地区：[1]江苏沿海地区农业科学研究所/农业农村部沿海盐碱地农业科学观测实验站,江苏盐城224001

出　　处：《作物学报》2024年第12期2971-2983,共13页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31501267);江苏省农业科技自主创新资金项目[CX(20)2065]资助。

摘　　要：研究低铁对Bt棉杀虫蛋白含量的影响及其生理和分子机制,可为Bt棉抗虫性安全表达提供理论参考。本文以转基因抗虫棉品种中棉50为试验材料,设置正常铁(CK,20.0µmol L^(–1))和低铁(LI,0.1µmol L^(–1))2个铁水平处理,采用水培法研究了低铁胁迫下Bt棉苗期Bt杀虫蛋白含量变化及氮代谢生理特征,通过转录组测序挖掘差异表达基因及其相关代谢途径,并采用qRT-PCR方法对测序结果进行验证。结果表明,与对照处理相比,低铁胁迫下Bt棉根和叶片中杀虫蛋白含量显著下降,且根中杀虫蛋白含量下降幅度更大。低铁胁迫降低了叶片中NH_(4)^(+)-N和NO_(3)^(–)-N含量,降低根和叶片中可溶性蛋白含量和全氮含量。根和叶片中硝酸还原酶、亚硝酸还原酶和谷氨酸合成酶活性的变化趋势与Bt杀虫蛋白含量表现一致。转录组结果表明,根和叶片中分别鉴定出11,661个和8972个差异表达基因,其中有1652个差异表达基因在根和叶片中均下调表达。GO注释表明,低铁处理根和叶片中差异基因的功能都主要富集于刺激反应、细胞壁、质膜、结合、氧化还原活性等。KEGG富集分析显示,苯丙烷类物质生物合成、苯丙氨酸代谢、半胱氨酸和蛋氨酸代谢、激素信号转导、玉米素生物合成、氮代谢、淀粉和蔗糖代谢、丙氨酸、天冬氨酸和谷氨酸代谢和酪氨酸代谢等途径在根和叶片中均发生显著变化。低铁胁迫下与氮素还原、同化途径相关的NR、NiR1和GLT1基因表达显著下调。以上结果表明,低铁胁迫会抑制Bt棉氮代谢相关基因的转录水平,减弱氮代谢的生理活性,抑制Bt杀虫蛋白的合成。To provide a theoretical basis for the safety of insecticidal efficiency in Bt cotton,this study investigated the effects of low iron on Bt insecticidal protein content and the physiological and molecular mechanisms in Bt cotton.Transgenic Bt cotton cultivar Zhongmian 50 was grown under low iron(LI,0.1µmol L^(-1) Fe(II)-EDTA)and normal iron(CK,20.0µmol L^(-1) Fe(II)-EDTA)conditions in hydroponic culture to assess the impact of low iron stress on Bt insecticidal protein content,activities of nitrogen metabolism-related enzymes,and substances in cotton seedlings.Differentially expressed genes(DEGs)were screened,and their relative expression patterns were analyzed by high-throughput sequencing,with results verified by qRT-PCR.The results showed that,compared with the control,the insecticidal protein contents significantly decreased under low iron conditions,with a more pronounced reduction in roots than in leaves.Iron deficiency decreased NH_(4)^(+)-N and NO_(3)^(-)-N levels in leaves and reduced soluble protein and nitrogen contents in both roots and leaves.Significant reductions in the activities of nitrate reductase(NR),nitrite reductase(NiR),and glutamine synthetase(GS)were observed when Bt insecticidal protein contents in roots and leaves were significantly reduced.High-throughput sequencing identified 11,661 DEGs in roots and 8972 in leaves,with 1652 genes down-regulated in both organs.GO annotation revealed that the functions of the differential genes in two organs were mainly concentrated in response to stimulus,cell wall,plasma membrane,binding,and catalytic activity.KEGG enrichment analysis demonstrated significant changes in phenylpropanoid biosynthesis,phenylalanine metabolism,cysteine and methionine metabolism,plant hormone signal transduction,zeatin biosynthesis,nitrogen metabolism,starch and sucrose metabolism,alanine,aspartate and glutamate metabolism,and tyrosine metabolism in both roots and leaves.Genes coding for NR,NiR1,and GLT1 related to the nitrogen reduction and assimilation pathway were si

关 键 词：BT棉 杀虫蛋白 低铁胁迫 转录组 

分 类 号：S562[农业科学—作物学]