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| 基于黏虫转录组的几丁质酶基因筛选和表达分析 |
| Screening and expression analysis of chitinase genes of Mythimna separata based on the transcriptome sequencing |
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| DOI： |
| 中文关键词: 黏虫 转录组 几丁质酶 基因表达 |
| 英文关键词:Mythimna separata (Walker) transcriptome chitinase gene expression |
| 基金项目:农业有害生物综合治理山西省重点实验室开放基金（YHSW2015001）；山西省科技重大专项计划“揭榜挂帅”项目课题（202101140601027）；国家自然科学基金杂粮专项（32241042）；国家自然基因重点项目（32130075）;杂粮种质资源创新与分子育种国家实验室（等）课题（202204010910001-34） |
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| 中文摘要: |
| 昆虫的几丁质酶对昆虫的生长发育致关重要，是生物农药的重要靶标。本研究使用高通量测序技术对迁飞性害虫黏虫Mythimna separata的中肠和表皮组织进行了转录组测序、序列组装、功能注释及差异表达基因分析。对转录组数据库中鉴定出的几丁质酶基因进行了理化性质的预测，包括cDNA长度、蛋白质分子量、氨基酸序列、等电点、不稳定系数、跨膜结构和蛋白结构域等。使用MEGA软件构建了黏虫和其他昆虫几丁质酶的系统进化树，并通过q-PCR验证了黏虫基因在不同组织和发育阶段的表达模式。通过中肠与表皮的转录组测序，获得了19.42 Gb的数据，在COG、GO、KEGG、KOG、Pfam、Swissprot、eggNOG、nr数据库注释到了25 236个Unigene；基因表达分析结果表明，中肠和表皮的差异表达基因共有3 137个，其中中肠高表达基因有1 872个，表皮高表达基因有1 265个。从转录组数据中鉴定出9个几丁质酶基因，其中7个是新的几丁质酶基因，这些基因的cDNA长度在1 362~9 816 bp，SMART结构预测表明几丁质酶含有1个或多个催化结构域。构建的系统进化树将昆虫几丁质酶基因分为9个亚家族。q-PCR结果表明〖STBX〗MsCht2、MsCht5、MsCht6、MsCht7、MsIDGF1在表皮中表达量较高，MsCht4、MsCht11和MsChi-H在中肠中表达量较高，与转录组数据一致；多数几丁质酶基因在蛹期或预蛹期表达量最高，而MsCht4在5龄期表达量最高，在蛹期表达量很低。黏虫几丁质酶基因表达上存在不同的差异，不同的几丁质酶基因可能具有不同的功能。本研究筛选出了7个新的几丁质酶基因，为黏虫的生物防治提供了新的靶标。研究结果为进一步研究黏虫几丁质酶的功能奠定了基础。 |
| 英文摘要: |
| Chitinase in insects is crucial for their growth and development and serves as an important target for biopesticides. This study aims to identify chitinase genes in armyworm Mythimna separata, a significant migratory pest. High-throughput sequencing technology was employed to perform transcriptome sequencing, sequence assembly, functional annotation, and differential gene expression 3 500 analysis on the midgut and epidermal tissues of armyworm. Chitinase gene sequences were identified from the transcriptome database, and the physicochemical properties such as cDNA length, protein molecular weight, amino acid sequence, isoelectric point, instability index, transmembrane structure, and protein domains of these genes were predicted. A phylogenetic tree of chitinases from armyworm and other insects was constructed using MEGA software, and the expression patterns of the genes at different tissues and developmental stages were verified by q-PCR. A total of 19.42 Gb of data were obtained from the transcriptomes sequence of the midgut and epidermis of armyworm, with 25 236 Unigenes annotated in COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG, and nr databases. Gene expression analysis revealed 3 137 differentially expressed genes between the midgut and epidermis, with 1 872 highly expressed genes in the midgut and 1 265 in the epidermis. 9 chitinase genes were identified from the transcriptome data, of which 7 genes were reported at first time, with cDNA lengths ranging from 1 362 to 9 816 bp. SMART structure prediction indicated that chitinases contain one or more catalytic domains. The constructed phylogenetic tree divided insect chitinase genes into nine subfamilies. q-PCR results showed that MsCht2, MsCht5, MsCht6, MsCht7, and MsIDGF1 were highly expressed in the epidermis, while MsCht4, MsCht11, and MsChi-H were highly expressed in the midgut, consistent with the transcriptome data. Most chitinase genes had the highest expression levels during the pupal or prepupal stages, on the other hand, MsCht4 exhibited the highest expression level during the 5th instar stage, while its expression level was considerably low during the pupa stage. There were distinct differences in gene expression between the midgut and epidermis of armyworm, indicating potential functional differences among different chitinase genes. The study identified 7 novel chitinase genes, providing new targets for the biological control of armyworm. The findings lay a foundation for further investigation into the functions of chitinases in armyworm. |
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