新麦草异戊烯基转移酶PjIPT基因的功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2024-0152

摘要/Abstract

摘要：

【目的】 异戊烯基转移酶（isopentenyl transferases, IPT）参与反式玉米素（trans-zeatin, tZ）的生物合成。tZ是调节植物生长发育和抵抗逆境胁迫的一种细胞分裂素类型，在促进芽生长和调控植物侧枝发育方面具有重要作用。探索IPT的功能，为后续新麦草产量性状改良提供了理论依据。【方法】 通过对新麦草IPT进行系统发育和多序列比对，探究其编码氨基酸序列的特征和同源性。通过花粉管通道法获得过表达PjIPT拟南芥（Arabidopsis thaliana）植株，并运用逆转录PCR（RT-PCR）和蛋白质印迹（Western blot, WB）对转基因植株进行验证，利用实时荧光定量逆转录PCR（RT-qPCR）对其组织表达模式进行分析，使用液质联用系统测定反式玉米素。【结果】 IPT在新麦草（Psathyrostachys juncea）和二粒小麦（Triticum dicoccoides）等麦类作物中具有高度同源性，且AtIPT9是PjIPT的同源蛋白。通过花粉管通道法获得过表达PjIPT拟南芥（Arabidopsis thaliana）植株，证明PjIPT上调植株分枝数。RT-PCR和蛋白质印迹分析显示，PjIPT在转录水平和蛋白水平均能够正常表达。利用不同部位组织RT-qPCR分析PjIPT的空间特异性表达，结果显示，PjIPT在拟南芥的分蘖节和叶中特异性表达。此外，反式玉米素的定量分析表明PjIPT通过参与tZ的生物合成去调控拟南芥植株分枝数。【结论】 PjIPT是上调植株分枝数的关键基因。

关键词: 新麦草, 异戊烯基转移酶, 反式玉米素, 分蘖, 功能验证

Abstract:

【Objective】 The isopentenyl transferase（IPT）enzyme plays a crucial role in the biosynthesis of trans-zeatin（tZ）, a cytokinin that regulates plant growth, development, and resistance to stress. It significantly contributes to bud growth promotion and regulation of lateral branch development in plants. Exploring the function of IPT provides a theoretical basis for the subsequent improvement of new wheat grass yield traits. 【Method】 Through phylogeny and multiple sequence alignment, the features and homology of the coding amino acid sequence were explored. PjIPT-overexpressed Arabidopsis thaliana plants were obtained by pollen tube passage, and the transgenic plants were verified by reverse PCR（RT-PCR）and protein blot（Western blot, WB）, their tissue expression patterns were analyzed by real-time quantitative reverse PCR（RT-qPCR）, and trans-zeatin was determined by liquid mass combination system. 【Result】 The IPT proteins in Psathyrostachys juncea with Triticum dicoccoides and other wheat crops had high homology, and AtIPT9 is a homologous protein of PjIPT. PjIPT-overexpressed in A. thaliana plants was achieved through the pollen tube pathway method, demonstrating an upregulation in the number of branches. RT-PCR and Western blot analysis confirmed the normal expressions of PjIPT at both transcriptional and protein levels. Spatial-specific expression analysis using RT-qPCR indicated that PjIPT demonstrated specific expression patterns in the tillering nodes and leaves of Arabidopsis. Furthermore, quantitative analysis of tZ revealed that PjIPT regulated the number of branches in Arabidopsis by participating in the biosynthesis of tZ. 【Conclusion】 PjIPT is a key gene involved in upregulating plant branching.

Key words: Psathyrostachys juncea, isopentenyl transferase, trans-zeatin, tillering, functional verification

任晓敏, 云岚, 艾芊, 赵乔. 新麦草异戊烯基转移酶PjIPT基因的功能验证[J]. 生物技术通报, 2024, 40(7): 207-215.

REN Xiao-min, YUN Lan, AI Qian, ZHAO Qiao. Functional Verification of Isopentenyl Transferases PjIPT Gene in Psathyrostachys juncea[J]. Biotechnology Bulletin, 2024, 40(7): 207-215.

图/表 7

表1 引物列表

Table 1 List of primers

名称 Name	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
RT-qPCR	GCAAGCCATGGAGTACCTGT	TCTCATTGCGGAACCAGGTC
Actin	AGTGGTCGTACAACCGGTATTGT	GAGGAAGAGCATACCCCTCGTA
RT-IPT	CTTTCGTGAGAGTGGGCAGT	GAAGCGTCAACCCACTGGTA

图1 反式玉米素的合成途径 ADP和ATP均为植物IPT酶的底物

Fig. 1 Synthesis pathway of trans-zeatin Both ADP and ATP are substrates of plant IPT enzymes

图2 基于IPT氨基酸序列的多序列比对及系统发育分析 A：不同物种IPT蛋白的多序列比对；B：不同物种IPT的系统进化树；Os：水稻；At：拟南芥

Fig. 2 Multiple sequence alignment and phylogenetic analysis based on IPT amino acid sequence A: Multiple sequence alignment of IPT proteins in different species. B: Phylogenetic tree of IPT in different species. Os: Oryza sativa; At: Arabidopsis thaliana

图3 阳性苗的RT-PCR（A）和Western blot（B）分析

Fig. 3 RT-PCR（A）and western blot（B）analysis of positive plants

图4 PjIPT在拟南芥不同组织中的表达 ****表示表达水平差异极显著（P<0.000 1）

Fig. 4 Expression analysis of PjIPT in the different tissues of A. thaliana **** indicates significantly different（P<0.000 1）

图5 35S:p1300-cYFP-PjIPT阳性植株表型分析 A：42日龄野生型与过表达PjIPT的代表性植株；B：42日龄Col-0和3个独立的35S:p1300-cYFP-PjIPT转基因拟南芥株系的分枝数和株高统计；***表示差异极显著（P<0.001）。下同

Fig. 5 Phenotypic analysis of 35S:p1300-cYFP-PjIPT positive plants A: Axillary branching of wild-type and overexpressed PjIPT in the representative plants described in B. B: Branch numbers and plant height in 42-day-old Col-0 and 3 independent 35S:p1300-cYFP-PjIPT transgenic Arabidopsis lines. ***: Extremely significant difference in（P <0.001）. The same below

图6 反式玉米素定量分析 A：不同样品中反式玉米素含量的分析；B：MRM色谱图；C：MRM质谱图

Fig. 6 Quantitative analysis of trans-zeatin A: Analysis of trans-zeatin content in different samples; B: MRM chromatograms; C: MRM mass spectrographs

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