意大利蜜蜂丝氨酸/苏氨酸蛋白激酶基因和全长转录本鉴定及验证

doi:10.16380/j.kcxb.2023.04.005

昆虫学报 ›› 2023, Vol. 66 ›› Issue (4): 478-485.doi: 10.16380/j.kcxb.2023.04.005

意大利蜜蜂丝氨酸/苏氨酸蛋白激酶基因和全长转录本鉴定及验证

范小雪^1,#, 张凯遥^1,#, 朱乐冉¹, 王紫馨¹, 张奎昊¹, 牛庆生³, 徐细建⁴, 骆群⁴, 陈大福^1,2,*, 郭睿^1,2,*

(1. 福建农林大学动物科学学院(蜂学学院), 福州 350002; 2. 福建省蜂疗研究所, 福州 350002; 3. 吉林省养蜂科学研究所, 吉林 132000; 4. 江西省养蜂研究所, 南昌 330000)

出版日期:2023-04-20 发布日期:2023-06-01

Identification and verification of genes and full-length transcripts of serine/threonine protein kinases in Apis mellifera ligustica

FAN Xiao-Xue^1,#, ZHANG Kai-Yao^1,#, ZHU Le-Ran¹, WANG Zi-Xin¹, ZHANG Kui-Hao¹, NIU Qing-Sheng³, XU Xi-Jian⁴, LUO Qun⁴, CHEN Da-Fu^1,2,*, GUO Rui^1,2,*

(1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Apitherapy Research Institute of Fujian Province, Fuzhou 350002, China; 3. Apiculture Science Institute of Jilin Province, Jilin 132000, China; 4. Apicultural Research Institute of Jiangxi Province, Nanchang 330000, China)

Online:2023-04-20 Published:2023-06-01

摘要/Abstract

摘要： 【目的】利用前期获得的高质量纳米孔长读段测序数据对意大利蜜蜂Apis mellifera ligustica的丝氨酸/苏氨酸蛋白激酶基因和全长转录本进行鉴定和分析，为深入开展功能研究提供参考信息和基础。【方法】基于前期获得的高质量意大利蜜蜂纳米孔长读段测序数据，通过Blast工具将意大利蜜蜂全长转录本比对Nr数据库筛选出丝氨酸/苏氨酸蛋白激酶基因和全长转录本；利用gffcompare软件将筛选出的丝氨酸/苏氨酸蛋白激酶全长转录本与西方蜜蜂A. mellifera参考基因组(Amel_HAv3.1)上注释的转录本进行比较，以鉴定未注释的新基因和新转录本；使用Astalavista软件鉴定丝氨酸/苏氨酸蛋白激酶基因的可变剪接(alternative splicing, AS)事件类型，采用IGV浏览器对剪接体的结构进行可视化，通过RT-PCR验证随机选取的6次AS事件的真实性。【结果】共鉴定到意大利蜜蜂丝氨酸/苏氨酸蛋白激酶71个基因和335条全长转录本，发掘出未注释的1个新基因和97条新转录本；共对14个已注释基因进行了结构优化，分别延伸了6个基因的5′端和8个基因的3′端。共鉴定到意大利蜜蜂丝氨酸/苏氨酸蛋白激酶7个基因的57次AS事件，包括40次外显子跳跃(exon skipping, ES)事件、 15次可变5′端剪接位点(alternative 5′splicing site, A5SS)事件和2次可变3′端剪接位点(alternative 3′splicing site, A3SS)事件。对随机选取的6次AS事件的RT-PCR结果表明，所有目的片段均符合预期大小，证实了AS事件的真实性。【结论】本研究系统鉴定了意大利蜜蜂丝氨酸/苏氨酸蛋白激酶基因和全长转录本，优化了西方蜜蜂参考基因组注释的丝氨酸/苏氨酸蛋白激酶的基因结构。

关键词: 意大利蜜蜂, 丝氨酸/苏氨酸蛋白激酶, 全长转录本, 第三代测序技术, 纳米孔测序

Abstract: 【Aim】To identify and analyze the genes and full-length transcripts of serine/threonine protein kinases of Apis mellifera ligustica using previously gained high-quality long-read nanopore sequencing data, and to provide reference information and bases for further functional study.【Methods】Based on the previously obtained high-quality long-read nanopore sequencing data of A. m. ligustica, the genes and full-length transcripts of serine/threonine protein kinases were screened from the Nr database by Blast. The screened full-length transcripts of serine/threonine protein kinases were compared with the annotated transcripts in the reference genome of A. mellifera (Amel_HAv3.1) using gffcompare software to identify the unannotated new genes and new transcripts. The types of alternative splicing (AS) events occurring in serine/threonine protein kinase genes were identified using Astalavista software. Visualization of the structure of spliceosomes was performed with IGV browser. RT-PCR was employed to confirm the authenticity of randomly selected six AS events.【Results】In total, 71 genes and 335 full-length transcripts of serine/threonine protein kinases of A. m. ligustica were identified, and one new gene and 97 new transcripts were discovered. The structure of 14 annotated genes was optimized, and the 5′ends of six genes and the 3′ends of eight genes were prolonged, respectively. A total of 57 AS events were identified in seven genes of serine/threonine protein kinases in A. m. ligustica, including 40 exon skipping (ES) events, 15 alternative 5′splicing site (A5SS) events and two alternative 3′splicing site (A3SS) events. RT-PCR results of randomly selected six AS events indicated that all of the target fragments were in accordance with the expected sizes, confirming the authenticity of AS events.【Conclusion】 Genes and full-length transcripts of serine/threonine protein kinases of A. m. ligustica were systematically identified and the structure of the serine/threonine protein kinase genes annotated in A. mellifera reference genome was optimized in this study.

Key words: Apis mellifera ligustica, serine/threonine protein kinase, full-length transcript, third generation sequencing technology, nanopore sequencing

范小雪, 张凯遥, 朱乐冉, 王紫馨, 张奎昊, 牛庆生, 徐细建, 骆群, 陈大福, 郭睿. 意大利蜜蜂丝氨酸/苏氨酸蛋白激酶基因和全长转录本鉴定及验证[J]. 昆虫学报, 2023, 66(4): 478-485.

FAN Xiao-Xue, ZHANG Kai-Yao, ZHU Le-Ran, WANG Zi-Xin, ZHANG Kui-Hao, NIU Qing-Sheng, XU Xi-Jian, LUO Qun, CHEN Da-Fu, GUO Rui. Identification and verification of genes and full-length transcripts of serine/threonine protein kinases in Apis mellifera ligustica[J]. Acta Entomologica Sinica, 2023, 66(4): 478-485.

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意大利蜜蜂丝氨酸/苏氨酸蛋白激酶基因和全长转录本鉴定及验证

Identification and verification of genes and full-length transcripts of serine/threonine protein kinases in Apis mellifera ligustica

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