Functional Verification of Isopentenyl Transferases PjIPT Gene in Psathyrostachys juncea

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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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