Cloning and Regulatory Activity Analysis of Soybean P34 Protein Gene Promoter

doi:10.7668/hbnxb.20195348

Abstract

Abstract:

Soybean P34 protein mainly exists in soybean seeds,and its upstream promoter was likely to regulate the high expression of downstream genes in seeds.In order to further study the tissue expression pattern of soybean P34 protein gene and the regulatory activity of soybean P34 protein gene promoter,qRT-PCR was used to detect the expression of soybean P34 protein gene in soybean tissues.The 5'upstream sequence of soybean P34 protein gene(GmP34P)was cloned.The transcription initiation sites and cis-elements were analyzed by bioinformatics.The expression vector was constructed and the tobacco was transformed by Agrobacterium-mediated leaf disk method to detect GUS expression in transgenic tobacco.The results showed that the expression of P34 protein gene in soybean seeds was significantly higher than that in roots,stems,leaves and flowers.The length of GmP34P sequence obtained by cloning was 1 380 bp.Predictive analysis showed that the transcriptional start site of this sequence was base A at position 1 342,and the sequence contained a variety of cis-acting elements related to high seed expression,such as RY element,Skn-1 motif,2S seed protbanapa,etc.The plant expression vector pCAM-GmP34P containing GUS gene driven by GmP34P promoter was obtained.The positive transgenic plants were screened by hygromycin,PCR and RT-PCR.The results showed that GUS gene expression was extremely significant in transgenic tobacco seeds compared with other tissues by qRT-PCR with positive pCAM-GmP34P transgenic tobacco plants.GUS histochemical staining showed that the GmP34P promoter could regulate the high expression of downstream GUS gene in seeds.

Key words: Soybean, P34 protein, Promoter, Bioinformatics, Transgenic tobacco

SANG Yingying, LI Shanshan, BAO Wei, XU Dong, ZHANG Xue, ZHAO Yan. Cloning and Regulatory Activity Analysis of Soybean P34 Protein Gene Promoter[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 22-28. doi: 10.7668/hbnxb.20195348.

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Figures/Tables 8

Tab.1 Primer sequences and uses

引物名称 Prime name	序列(5'—3') Sequence (5'—3')	引物用途 Primer use
A₁	GGAAGGCTTTCTTGCATTGGTA	大豆中qRT-PCR检测(内参基因)
A₂	AGTGGCATCCTGGTACTGC
B₁	TGTGACCATCCACCTGCATCATG	大豆中qRT-PCR检测 (P34蛋白基因)
B₂	CAACCGCTTCCACAGCCC
C₁	ATGAGAGAGTGCATATCGAT	烟草中qRT-PCR检测(内参基因)
C₂	TTCACTGAAGAAGGTGTTGAA
D₁	GATCGCGAAAACTGTGGAAT	烟草中qRT-PCR检测(GUS基因)
D₂	TAATGAGTGACCGCATCGAA
E₁	TGAGTTGTTTCAGGTTCC	GmP34P片段的PCR扩增
E₂	GGGCCATGGAACTTGGTGGAAGAATTTTATG
F₁	GTAGAAACCCCAACCCGTGAA	转基因烟草RT-PCR检测
F₂	TGAGCGTCGCAGAACATTACAT

Fig.1 Expression analysis of soybean P34 protein gene in soybean tissues Different capital letters indicate significant differences in different tissues(P<0.01).The same as Fig.6.

Fig.2 PCR amplification of GmP34P promoter and identification of pMD18-T-GmP34P plasmid by double enzyme digestion M.DL2000 Marker;A:1.PCR amplification of GmP34P promoter fragment;B:1.pMD18-T-GmP34P plasmid double enzyme digestion.

Fig.3 Analysis of cis-acting elements of GmP34P

Fig.4 Identification of pCAM-GmP34P plasmid by double enzyme digestion and PCR of Agrobacterium solution M.DL2000 Marker;A:1.Results of double enzyme digestion of pCAM-GmP34P plasmid;B:1.PCR of Agrobacterium solution.

Fig.5 PCR and RT-PCR identification of T₁transgenic tobacco plants with pCAM-GmP34P M.DL2000 Marker;+.Positive control;-.Negative control;A:1—6.PCR identification of transgenic tobacco plants;B:1—6.RT-PCR identification of transgenic tobacco plants.

Fig.6 GUS gene expression in tissues of transgenic pCAM-GmP34P tobacco

Fig.7 GUS histochemical staining analysis WT.Wild type tobacco seeds;GmP34P.Tobacco seeds transformed with pCAM-GmP34P expression vector;CaMV35S.Tobacco seeds transformed with pCAMBIA1301 expression vector.

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