枳PtMLP1启动子的克隆和表达分析

doi:10.3864/j.issn.0578-1752.2023.24.009

摘要/Abstract

摘要：

【目的】基因工程是柑橘品种改良的一种重要手段。本研究基于枳根消减文库中主要乳胶蛋白基因PtMLP1片段，克隆根特异启动子序列，为研究外源基因在柑橘根组织的特异表达奠定基础。【方法】同源克隆PtMLP1及启动子序列。利用ExPASy、PSIPRED、SWISS-MODEL等在线软件对PtMLP1编码蛋白的理化特征、二级结构和三级结构进行生物信息学分析，利用PlantCARE数据库对PtMLP1启动子的顺式作用元件进行预测。实时荧光定量PCR法对PtMLP1在不同树龄枳根和叶中的表达进行分析。构建PtMLP1启动子与GUS标记基因的融合载体，利用根癌农杆菌转化法转化枳上胚轴，GUS染色观察标记基因的表达部位。【结果】枳PtMLP1含2个外显子和1个内含子，开放阅读框长471 bp。PtMLP1蛋白由156个氨基酸组成，分子量17.63 kDa，等电点5.49，含Bet v I功能域。其二级结构含3个α-螺旋和7个β-折叠，三级结构包含一个保守疏水基结合位点和一个富含甘氨酸的回环结构。5′端1 666 bp的上游调控序列不仅有TATA-box、CAAT-box等启动子结构的核心元件，还具有多个根组织特异表达元件，以及TGACG-motif、P-box和ABRE等激素应答相关的顺式作用元件。3′端非翻译区具有加尾信号AATAAA。该基因在1月龄苗、6月龄苗、20年生成年枳根中的表达量分别是叶中的46.34、74.82、110.25倍。构建启动子的融合表达载体pBI121-ProPtMLP1::GUS，获得枳转基因植株。PtMLP1启动子驱动GUS在转基因枳幼苗根中特异表达，GUS在3个转基因枳株系的根中表达量分别为叶中表达量的124.78、11.53和7.76倍。【结论】获得柑橘主要乳胶蛋白PtMLP1及启动子序列，该启动子可驱动标记基因在柑橘根组织特异表达。

关键词: 枳, 主要乳胶蛋白, 根特异性启动子, GUS

Abstract:

【Objective】Genetic transformation plays a significant role in exploring gene function and improving traits in citrus. Tissue-specific promoters is a key to regulate the expression of transgenes in particular tissues. Here, expression characteristics of the PtMLP1 promoter, isolated from the root subtractive library of Poncirus trifoliata, was thoroughly examined, which could lay a foundation for the specific expression of exogenous genes in citrus root tissue. 【Method】The complete sequence of PtMLP1 gene was cloned by PCR using DNA as a template. The physiochemical attributes, secondary and tertiary structures of PtMLP1 protein were predicted by ExPASy, PSIPRED, and SWISS-MODEL tools. Cis-acting elements in PtMLP1 promoter were predicted by PlantCARE. The expression pattern of PtMLP1 in P. trifoliata trees of diverse ages was examined by employing real-time qPCR. Furthermore, to investigate the tissue-specific expression of the PtMLP1 promoter in citrus, a pBI121-ProPtMLP1::GUS plasmid, in which GUS expression was controlled by the PtMLP1 promoter, was constructed and then introduced into P. trifoliata through Agrobacterium-mediated hypocotyl transformation. 【Result】PtMLP1 consisted of two exons and one intron, which possessed a 471 bp open reading frame encoding a protein with 156 amino acid residues. This protein had a molecular weight of 17.63 kilodaltons with an isoelectric point of 5.49 and contained a Bet v I functional domain in its primary structure. Moreover, the secondary structure of PtMLP1 contained three α-helices and seven β-folds, while its tertiary structure had a conserved hydrophobic binding site and a cyclic domain, which was rich in glycine. The PtMLP1 promoter was 1 666 bp long. Multiple root-specific expression elements, phytohormone response elements (such as the TGACG motif, P-box, and ABRE), and the TATA box and CAAT box core elements were predicted in the promoter. Additionally, the 3-terminal untranslated region of PtMLP1 was predicted to contain a poly (A) signal AATAAA. Notably, the expression of PtMLP1 was significantly higher in the roots of 1-month, 6-month, and 20-year-old P. trifoliata, with fold changes of 46.34, 74.82, and 110.25, respectively, compared with those in leaves. GUS expression analysis of pBI121-ProPtMLP1::GUS transgenic plants showed that PtMLP1 promoter exhibited specific and high expression in roots, and its expression levels were 7.76 to 124.78 times of that in the leaves. 【Conclusion】The sequences of the PtMLP1 gene and its promoter were successfully obtained, and the promoter demonstrated the ability to drive specific expression of GUS gene in citrus roots.

Key words: Poncirus trifoliata, major latex protein, root-specific promoter, GUS

姚利晓, 苏娟, 郭兴茹, 李凤龙, 何永睿, 邹修平, 陈善春. 枳PtMLP1启动子的克隆和表达分析[J]. 中国农业科学, 2023, 56(24): 4906-4915.

YAO LiXiao, SU Juan, GUO XingRu, LI FengLong, HE YongRui, ZOU XiuPing, CHEN ShanChun. Cloning and Expression Analysis of PtMLP1 Promoter in Poncirus trifoliata[J]. Scientia Agricultura Sinica, 2023, 56(24): 4906-4915.

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引物名称 Primer name	引物序列 Primer sequence	作用 Function
Con2F	5′-ACCGTTCAAGCCATTCCGAAGG-3′	PtMLP1实时荧光定量PCR Real time qPCR for PtMLP
Con2R	5′-TGGGGGATGATGGGCTTCGAT-3′	PtMLP1实时荧光定量PCR Real time qPCR for PtMLP
qAct1	5′-CTGCTGGAATTCACGAAACA-3′	Actin实时荧光定量PCR Real time qPCR for Actin
qAct2	5′-GCCACCACCTTGATCTTCAT-3′	Actin实时荧光定量PCR Real time qPCR for Actin
GUSF	5′-CGTTAGCCGGGCTGCACTCA-3′	GUS实时荧光定量PCR Real time qPCR for GUS
GUSR	5′-CCGCCAACGCGCAATATGCC-3′	GUS实时荧光定量PCR Real time qPCR for GUS
2IS	5′-GACCCTATCTGGTCAGTTGGAGAC-3′	克隆PtMLP1内含子 Cloning the intron of PtMLP1
2IR	5′-CACCCTTCGGAATGGCTTG-3′	克隆PtMLP1内含子 Cloning the intron of PtMLP1
2PaS2	5′-TATaagcttATCTTCGGCTGTAGGCCACATCA-3′	PtMLP1启动子克隆 Cloning the promoter of PtMLP1
2PaR1	5′-GCGtctagaGTTTTTAGCGGATTAAAATA-3′	PtMLP1启动子克隆 Cloning the promoter of PtMLP1