孝顺竹中笋箨衰老相关WRKY转录因子的鉴定与分析

却枫; 刘庆楠; 查若飞; 魏强

doi:10.12302/j.issn.1000-2006.202206049

却枫, 刘庆楠, 查若飞, 魏强

南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (6) : 113-123.

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南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (6) : 113-123. DOI: 10.12302/j.issn.1000-2006.202206049

研究论文

孝顺竹中笋箨衰老相关WRKY转录因子的鉴定与分析

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Identification and analysis of senescence related WRKYs in sheath of Bambusa multiplex

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摘要

【目的】孝顺竹(Bambusa multipex)笋箨中含有大量的叶绿体,具有进行光合作用的功能,是竹秆快速生长时期的一个重要碳源。笋箨与所附着节间是一种“源-库”关系,其衰老对节间的伸长生长具有重要影响。通过鉴定和分析孝顺竹笋箨中衰老相关的WRKY转录因子,为研究笋箨的衰老机制和调控机制奠定基础。【方法】基于孝顺竹笋箨3代全长转录组数据库,使用HMMER软件进行WRKY转录因子的初步搜索,之后通过保守结构域分析和去冗余获得候选基因。与拟南芥(Arabidopsis thaliana)中已验证过的参与叶片衰老调控的WRKY转录因子进行同源性比对分析,以预测孝顺竹WRKY转录因子家族中参与衰老调控的成员。基于不同发育时期笋箨的2代转录组数据,分析候选BmWRKYs在笋箨发育不同阶段的表达模式以进行功能预测。通过检测叶绿素含量和膜离子渗透率对不同长度第3节间笋箨的发育状态进行鉴别,并进行实时荧光定量PCR实验对部分候选基因在笋箨不同发育时期的表达水平进行检测,以对WRKY基因家族中与笋箨衰老相关的成员进行进一步验证。【结果】在孝顺竹笋箨中共鉴定到83个蛋白质序列长度为119~1 751个氨基酸的WRKY转录因子。基于蛋白质结构和系统发育分析,将83个BmWRKYs分为Ⅰ、Ⅱ a—e和Ⅲ 3大组,其中被鉴定为Ⅰ类的BmWRKY转录因子最多,为39个;Ⅱ a、Ⅱ b、Ⅱ c、Ⅱ d、Ⅱ e亚家族分别有2、16、11、12和2个BmWRKY成员;仅有1个BmWRKY被鉴定为Ⅲ类WRKY转录因子。此外,通过与拟南芥基因组中已鉴定的衰老相关WRKY进行同源比对分析,共获得31个与笋箨衰老相关的候选基因BmWRKYs。基于候选基因BmWRKYs在孝顺竹笋箨不同发育时期的表达模式,将31个候选BmWRKYs分为a、b、c、d 4种类型。a类包括BmWRKY59、BmWRKY75、BmWRKY6、BmWRKY4、BmWRKY41、BmWRKY51、BmWRKY26、BmWRKY48、BmWRKY53、BmWRKY36、BmWRKY42、BmWRKY18和BmWRKY10;b类包括BmWRKY2、BmWRKY74、BmWRKY38、BmWRKY76、BmWRKY30、BmWRKY43、BmWRKY69和BmWRKY72;c类包括BmWRKY15、BmWRKY68、BmWRKY28、BmWRKY12和BmWRKY7;d类包括BmWRKY3、BmWRKY21、BmWRKY1、BmWRKY25和BmWRKY5。孝顺竹不同长度第3节间所附着的笋箨具有不同的发育状态,当第3节间长度在20~25 cm时叶绿素已开始出现降解。随着第3节间伸长生长减缓程度的增加,笋箨的衰老程度也在加剧,当节间长度为30 cm左右时,笋箨已明显衰老。部分BmWRKYs在孝顺竹不同发育时期笋箨中的表达水平表明,a类BmWRKYs中,BmWRKY4、BmWRKY48和BmWRKY53可能是笋箨衰老的正调控因子,而d类中的BmWRKY21可能是孝顺竹笋箨衰老的负调控因子,并且在笋箨发育早期起作用。【结论】笋箨是孝顺竹节间快速生长阶段的重要碳源,其衰老涉及营养物质的再动员。对笋箨衰老调控的研究是节间快速伸长机制研究的一个重要切入点。本研究鉴定得到多个在孝顺竹笋箨衰老进程中具有重要潜在作用的BmWRKYs,研究结果将为今后深入分析WRKY转录因子调控笋箨衰老的分子机制奠定基础。

Abstract

【Objective】The sheaths of Bambusa multiplex, containing numerous chloroplasts capable of photosynthesis, serve as a significant carbon source during the fast growth stage of B. multiplex. The sheath senescence profoundly impacts the growth of the culm elongation. While the WRKY gene family is known for its pivotal role in regulating various plant developmental processes, limited information is available regarding its role in B. multiplex. Therefore, this work aimed to identify and analyze the senescence-related WRKY genes in the sheath of B. multiplex. 【Method】Based on the full-length transcripts of the B. multiplex sheath, HMMER software was adopted to the primary search of WRKY transcription factors. Then, the candidate WRKYs in the sheath transcripts were obtained by conservation domain analysis and with redundancy removal. To predict the candidate BmWRKYs involved in regulating the sheath senescence, the phylogenetic analysis between BmWRKYs and the senescence-related AtWRKYs (Arabidopsis) genes was determined. Then, based on second-generation transcriptome data obtained from different developmental stages of the sheaths of B. multiplex, the expression patterns of BmWRKYs at different developmental stages of sheaths were analyzed for functional prediction. In addition, the developmental stages of sheaths that attached to the third internode with different lengths were identified by detecting chlorophyll content and membrane ion leakage. The expression profiles of BmWRKYs in the sheath with different developmental stages were also detected by qRT-PCR to further determine the roles of BmWRKYs during the sheath senescence. 【Result】A total of 83 BmWRKYs were identified, with protein lengths ranging from 119 to 1 751. Based on the protein structure and phylogenetic analysis, these 83 BmWRKYs were classified into three subgroups: Ⅰ, Ⅱ a-e, and Ⅲ. Specifically, 39 BmWRKYs belonged to subgroup I, while 43 BmWRKYs belonged to subgroup Ⅱ a-e. Within the subgroup Ⅱ a-e, 2, 16, 11, 12, and 2 BmWRKYs were categorized into subgroups Ⅱ a, Ⅱ b, Ⅱ c, Ⅱ d, and Ⅱ e, respectively. Only one BmWRKY was classified into subgroup Ⅲ. Additionally, 31 BmWRKYs likely involved in regulating sheath senescence were identified through homologous analysis with senescence-related AtWRKYs. These 31 BmWRKYs were further categorized into four types (a, b, c and d) based on their expression patterns during different stages of the sheath senescence. BmWRKY59, BmWRKY76, BmWRKY6, BmWRKY4, BmWRKY41, BmWRKY51, BmWRKY26, BmWRKY48, BmWRKY53, BmWRKY36, BmWRKY42, BmWRKY18 and BmWRKY10 were a-type. BmWRKY2, BmWRKY74, BmWRKY38, BmWRKY76, BmWRKY30, BmWRKY43, BmWRKY69 and BmWRKY72 were b-type. BmWRKY15, BmWRKY68, BmWRKY28, BmWRKY12 and BmWRKY7 were c-type. BmWRKY3, BmWRKY21, BmWRKY1, BmWRKY25 and BmWRKY5 were d-type. Sheaths that attached to internodes with different length had different developmental stages. When the length of the third internode was between 20-25 cm, chlorophyll began to degrade. When the internode length was about 30 cm, the sheath had significant senescence. Based on these observations, few BmWRKYs were further selected to measure their expression profiles in the sheath at different developmental stages. The results suggested that BmWRKY4, BmWRKY48 and BmWRKY53 (a-type) appeared to be positive regulators of sheath senescence, while BmWRKY21 (d-type) was a potential negative regulator, acting during the early stages of sheath senescence. 【Conclusion】The sheaths play a crucial role as carbon sources during the rapid growth stage of B. multiplex internodes, and their senescence involves nutrient mobilization. Investigating sheath senescence regulation serves as a key entry point for understanding the mechanism of rapid internode elongation. This study identified multiple BmWRKYs with significant potential roles in the sheath senescence process. These results lay the foundation for further analysis of the molecular mechanism of WRKY transcription factors regulating the sheath senescence in the future.

导出引用

却枫, 刘庆楠, 查若飞, 等. 孝顺竹中笋箨衰老相关WRKY转录因子的鉴定与分析[J]. 南京林业大学学报（自然科学版）. 2023, 47(6): 113-123 https://doi.org/10.12302/j.issn.1000-2006.202206049

QUE Feng, LIU Qingnan, ZHA Ruofei, et al. Identification and analysis of senescence related WRKYs in sheath of Bambusa multiplex[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2023, 47(6): 113-123 https://doi.org/10.12302/j.issn.1000-2006.202206049

中图分类号： Q786;Q781

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