褐飞虱中富含丝氨酸/精氨酸的可变剪接因子特性和生物学功能研究

doi:10.16819/j.1001-7216.2025.240804

摘要/Abstract

摘要：

【目的】SR蛋白(serine/arginine-rich protein)是一类富含丝氨酸/精氨酸的可变剪接因子，在褐飞虱体内对基因的剪接过程起调控作用。本研究通过克隆褐飞虱(Nilaparvata lugens)的5个NlSR基因并鉴定了其分子特性、表达模式和生物学功能，为褐飞虱的防治提供新的思路。【方法】基于褐飞虱基因组数据，利用PCR技术克隆了5个NlSR基因cDNA序列，并利用生物信息学手段分析其序列特征；利用qRT-PCR技术检测它们在不同发育阶段 (卵、1 - 5龄若虫和雌雄成虫)和组织(唾液腺、体壁、脂肪体、卵巢和中肠)中的表达特征；并测定干扰后的基因表达及褐飞虱的存活率、蜜露量和体质量变化。【结果】成功克隆了5个NlSR基因的cDNA序列，分别命名为NlSRSF1、NlSRSF2.1、NlSRSF2.2、NlSRSF7.1和NlSRSF7.2。根据进化树分类，这些基因被分为3个亚家族：SRSF1、SRSF2和SRSF7。NlSRs基因的开放阅读框长度在495 bp到508 bp之间，编码的氨基酸数目介于164 aa到235 aa，预测分子量在19.54 kD到26.76 kD之间，等电点在9.53到11.83之间，均为亲水性碱性蛋白，不稳定指数在63.55到131.97之间。编码的蛋白含有N端的RRM结构域(RNA recognition motif)和C端的RS结构域(Arginine/serine-rich domain)，NlSRSF7.1和NlSRSF7.2还含有锌指(ZnF_C2HC)结构域。NlSR基因在褐飞虱的唾液腺、体壁、脂肪体、卵巢和中肠等组织中均有表达，其中NlSRSF1和NlSRSF2.1在唾液腺中的相对表达量较高；NlSRSF1主要在卵和1龄若虫中表达，其他基因主要在成虫中表达。RNAi实验结果表明，与对照组dsGFP相比，干扰NlSRSF1、NlSRSF2.1和NlSRSF7.2显著降低褐飞虱的存活率，干扰NlSRSF2.2和NlSRSF7.1对存活率无显著影响，干扰所有NlSR基因均显著降低了褐飞虱的蜜露量和体质量增加量。【结论】本研究克隆了褐飞虱的5个NlSR基因，分析了其序列和表达特征，并通过干扰确定它们对褐飞虱生命活动的影响，为深入研究褐飞虱NlSRs的生物学功能奠定了基础。

关键词: 褐飞虱, SR蛋白, 可变剪接, RNAi

Abstract:

【Objective】 Serine/arginine-rich (SR) proteins are a class of alternative splicing factors that regulate gene splicing in the brown planthopper, Nilaparvata lugens. This study aimed to clone five NlSR genes from N. lugens and identify their molecular characteristics, expression patterns, and biological functions, providing new insights for controlling brown planthoppers. 【Method】Based on the genomic data of N. lugens, the cDNA sequences of five NlSR genes were cloned using RT-PCR. Their sequence characteristics were analyzed. The expression profiles of NlSRs in different developmental stages (egg, 1st-5th instar nymphs, and male and female adults) and tissues (salivary glands, integument, fat body, ovaries, and midgut) of N. lugens were detected using qRT-PCR. RNA interference (RNAi) technology was utilized to interfere with the relative expression levels of the NlSR genes. The expression of NlSRs, survival rates, honeydew production, and body weight gains of N. lugens after RNAi were measured. 【Results】Five cDNA sequences of NlSR genes were cloned, which were designated as NlSRSF1, NlSRSF2.1, NlSRSF2.2, NlSRSF7.1, and NlSRSF7.2. Based on phylogenetic tree analysis, the deduced proteins were classified into three subfamilies: SRSF1, SRSF2, and SRSF7. The open reading frames of the NlSR genes ranged from 495 to 508 bp, encoding proteins with 164－235 amino acids. The predicted molecular weights ranged from 19.54 to 26.76 kD, with isoelectric points ranging from 9.53 to 11.83. These proteins were hydrophilic and alkaline, with instability indices ranging from 63.55 to 131.97. The encoded proteins contained an N-terminal RNA recognition motif (RRM) and a C-terminal arginine/serine-rich domain (RS). NlSRSF7.1 and NlSRSF7.2 also contained a ZnF_C2HC domain. NlSRs were expressed in various tissues of N. lugens, including salivary glands, integument, fat body, ovaries, and midgut. Among them, NlSRSF1 and NlSRSF2.1 exhibited high relative expression levels in salivary glands. NlSRSF1 was primarily expressed in eggs and 1st instar nymphs, while the remaining NlSR genes were predominantly expressed in adults. RNAi experiments showed that targeting NlSRSF1, NlSRSF2.1, and NlSRSF7.2 significantly reduced the survival rate of N. lugens compared to the control group (dsGFP). However, interference with NlSRSF2.2 and NlSRSF7.1 did not significantly affect survival rates. Interference with all NlSRs significantly reduced honeydew production and body weight gains in N. lugens. 【Conclusion】This study successfully cloned five NlSR genes from N. lugens, analyzed their sequence and expression characteristics, and determined their effects on the life activities of N. lugens through interference. This provides a foundation for further investigation into the biological functions of NlSRs in N. lugens.

Key words: Nilaparvata lugens, SR protein, alternative splicing, RNAi

贾毅帆, 王新峰, 王雅宣, 刘芳, 肖晶, 魏琪, 傅强, 万品俊. 褐飞虱中富含丝氨酸/精氨酸的可变剪接因子特性和生物学功能研究[J]. 中国水稻科学, 2025, 39(2): 277-286.

JIA Yifan, WANG Xinfeng, WANG Yaxuan, LIU Fang, XIAO Jing, WEI Qi, FU Qiang, WAN Pinjun. Molecular Characterization and Biological Function of Serine/Arginine-rich Alternative Splicing Factors in Nilaparvata lugens (Hemiptera:Delphacidae)[J]. Chinese Journal OF Rice Science, 2025, 39(2): 277-286.

图/表 7

表1 本研究所用的扩增引物、表达引物和双链合成引物

Table 1. Primers for amplification, mRNA expression and dsRNA synthesis in this study

引物名称 Primer	上游引物序列(5′-3′) Upstream primer sequences (5′-3′)	下游引物序列(5′-3′) Downstream primer sequences (5′-3′)	Use 用途
cNlSRSF1	AAGACGACGCCTTGCCT	AGCTGTGTGTTGCTCCTTAATT	克隆 Clone
cNlSRSF2.1	TCCACAACTTCACATAAGTTCG	TTAGGCTTGGTAGCCAGCA
cNlSRSF2.2	GCATCTGTTTCTTGGCGG	TGAGCAGTATTGGTCGCCT
cNlSRSF7.1	TCGGTAGTAGCCTAAGCAGTCA	ACGTGGGTATCAAATAATTAACAAA
cNlSRSF7.2	CATGTAATTTCTGCTGGTGTTG	GACTTTGTTGTGTGATGTGAGG
qNlSRSF1	GATATCGCGACAGCGAGGAC	CACCGGAGAGTAAACCGGAC	qPCR
qNlSRSF2.1	GCGGTCAGACAGCAAGAGTT	GAGTGCGACCTTGACTTGGA
qNlSRSF2.2	GAGCTGAAAGTTCAGATGGC	TGATGCGAGTGATGGCGAC
qNlSRSF7.1	CGCGGTCACTACGCAAGGAAC	GGACGACCTTGACCGTGAC
qNlSRSF7.2	AGGTCACGCGATAGACGTTC	CGTTGCCATTTCTCTCGGGT
qNlRPS15	TAAAAATGGCAGACGAAGAGCCCAA	TTCCACGGTTGAAACGTCTGCG
dsNlSRSF1	T7-TTGAAGAACCGAAGAGGCCC	T7-TAAATCCTGCCAGCTTCCCG	RNAi
dsNlSRSF2.1	T7-GTCCGTCTAAAAGGCGTCCA	T7-ATGAGTTACGGACGCCCACC
dsNlSRSF2.2	T7-GTTTTGGAAGACCACCCCCT	T7-TCGGCATCACGCTTGTCATA
dsNlSRSF7.1	T7-TGCGTAGTGACCGCGTTTAT	T7-AATCAGGAACGTGTGGGTGG
dsNlSRSF7.2	T7-ATACAGGGACTCAGGCAACC	T7-GCTACGCCCATCTAGACCAC
dsGFP	T7-CCTGAAGTTCATCTGCACCAC	T7-TGATGCCGTTCTTCTGCTTGT

图1 五个NlSR的多序列比对和功能结构域氨基酸位置显示在右边，预测的RRM结构域、RS结构域和ZnF_C2HC结构域分别用框线标记。

Fig. 1. Alignment of amino acid residues of SRs in Nilaparavata lugens Amino acid positions are indicated on the right. The predicted RRM domain, RS domain, and ZnF_C2HC domain are highlighted with boxes, respectively.

图2 褐飞虱SR与其他昆虫同源序列的系统进化树 Tc: 赤拟谷盗; Ap: 豌豆蚜; Dc: 柑橘木虱; Ha: 棉铃虫; Sl: 斜纹夜蛾; Of: 亚洲玉米螟。系统发育构建使用邻接法；不同阴影代表不同分组。

Fig. 2. Phylogenetic tree of NlSRs in Nilaparvata lugens and homologs from other insects Tc, Tribolium castaneum；Ap, Acyrthosiphon pisum；Dc, Diaphorina citri；Ha, Helicoverpa armigera；Sl, Spodoptera litura；Of, Ostrinia furnacalis. The phylogenetic tree was constructed using the neighbor-joining method, and different groups are represented by shaded areas.

图3 褐飞虱不同组织间NlSR基因的相对表达量 qPCR均以NlRPS15基因为内参，所有数据为各个组织褐飞虱NlSR基因的相对表达量±标准误。图中数据采用单因素方差分析（ANOVA）和Tukey法进行多重比较检验，数据柱上标的不同字母表示组间存在显著性差异（P < 0.05）。

Fig. 3. Relative expression levels of NlSRs in various tissues of Nilaparavata lugens The expression levels of NlSR genes in various tissues of Nilaparvata lugens were determined by qPCR, using the NlRPS15 gene as an internal reference. The bars represent 2−△△CT values (± SE), normalized to the geometric mean of housekeeping gene expression. Different letters indicate significant differences at P value < 0.05, as analyzed by ANOVA and Turkey’s test.

图4 NlSRs基因在褐飞虱不同发育时期的表达 qPCR均以NlRPS15基因为内参，所有数据为各个龄期褐飞虱NlSR基因的相对表达量±标准误。图中数据采用ANOVA和Turkey法分析检验，数据柱上标的不同字母代表数字之间的显著性差异（P < 0.05）。

Fig. 4. Relative expression levels of NlSRs in different developmental stages of Nilaparavata lugens The expression levels of NlSR genes in different development stages of Nilaparavata lugens were determined by qPCR, using the NlRPS15 gene as an internal reference. The bars represent 2-△△CT values ( ± SE) normalized to the geometric mean of housekeeping gene expression. Different letters indicate a significant difference at P value < 0.05, as analyzed by ANOVA and Turkey’s test.

图5 dsRNA注射后第3天NlSR相对表达量变化 qPCR均以NlRPS15基因为内参，所有数据为各处理组NlSRs基因的相对表达量 ± 标准误。图中数据采用非配对t检验法分析，*代表P < 0.05，**代表P < 0.01，***代表P < 0.001。

Fig. 5. Relative expression level of NlSRs on the 3rd day after injection with dsRNA in Nilaparavata lugens Utilizing the brown planthopper RPS15 gene as an internal reference for normalization, the relative expression levels of NlSR were determined in each treatment. Different symbols indicate significant differences, as analyzed by the Student’s t test. * P < 0.05, ** P < 0.01, and *** P < 0.001.

图6 注射dsNlSRSF后褐飞虱4龄若虫的存活率(A)、雌成虫的蜜露量(B)和体质量增量(C) 以dsGFP为对照组，B和C图中的数据为平均值 ± 标准误，柱上不同字母表示基因表达量经Tukey HSD检验存在显著差异（P < 0.05）。A图中数据采用Kaplan-Meier法计算，并采用Log-rank检验进行比较。

Fig. 6. Survival rate (A) of the 4th instar nymphs and the honeydew amount (B) and body weight gain (C) of Nilaparvata lugens after dsRNA injection The brown planthopper injected with dsGFP were used as controls. Data in Figs. B and C are mean ± SE. Different letters above bars indicate significant differences (P < 0.05) in the gene expression levels, as analyzed by ANOVA and the Tukey’s HSD test. Data in Fig. A were calculated by the Kaplan-Meier method and compared by the log-rank test.

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