大豆P34蛋白基因启动子克隆及其调控活性分析

doi:10.7668/hbnxb.20195348

摘要/Abstract

摘要：

大豆P34蛋白主要存在于种子中,其上游启动子很可能具有调控下游基因在种子中高表达的特性。为进一步研究大豆P34蛋白基因的组织表达模式及其启动子的调控活性,通过qRT-PCR方法检测大豆P34蛋白基因在大豆不同组织中的表达情况;克隆大豆P34蛋白基因5'端上游序列(GmP34P),生物信息学分析其转录起始位点和顺式元件;构建表达载体,利用农杆菌介导的叶盘法转化烟草,检测转基因烟草中GUS的表达。结果表明,P34蛋白基因在大豆种子中的表达量极显著高于根、茎、叶和花的表达量;克隆获得GmP34P序列长度为1 380 bp,预测分析表明,该序列的转录起始位点为第1 342位上的碱基A,序列中含有多种与种子高表达相关的顺式作用元件,如RY element、Skn-1 motif、2S seed protbanapa等;获得含有GmP34P启动子驱动GUS基因的植物表达载体pCAM-GmP34P;通过潮霉素、PCR及RT-PCR筛选阳性转基因植株;对pCAM-GmP34P阳性转基因烟草植株,进行qRT-PCR检测,结果显示,相对于其他组织,GUS基因在转基因烟草种子中的表达量达到极显著差异;GUS组织化学染色结果显示,GmP34P启动子能够调控下游GUS基因在种子中高表达。

关键词: 大豆, P34蛋白, 启动子, 生物信息学分析, 转基因烟草

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

桑莹莹, 李珊珊, 鲍薇, 徐东, 张雪, 赵艳. 大豆P34蛋白基因启动子克隆及其调控活性分析[J]. 华北农学报, 2025, 40(1): 22-28. doi: 10.7668/hbnxb.20195348.

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.

http://www.hbnxb.net/CN/Y2025/V40/I1/22

图/表 8

表1 引物序列及用途

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

图1 大豆P34蛋白基因在大豆各组织中的表达不同大写字母表示不同组织间的差异极显著(P<0.01)。图6同。

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.

图2 GmP34P启动子的PCR扩增及pMD18-T-GmP34P质粒的双酶切鉴定 M.DL2000 Marker;A:1.GmP34P启动子PCR扩增片段;B:1.pMD18-T-GmP34P质粒双酶切结果。

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.

图3 GmP34P的顺式作用元件分析

Fig.3 Analysis of cis-acting elements of GmP34P

图4 pCAM-GmP34P质粒的双酶切鉴定及农杆菌菌液PCR鉴定 M.DL2000 Marker;A:1.pCAM-GmP34P 质粒的双酶切结果;B:1.农杆菌菌液PCR。

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.

图5 T₁ pCAM-GmP34P转基因烟草植株的PCR鉴定和RT-PCR鉴定 M.DL2000 Marker;+.阳性对照;-.阴性对照;A:1—6.转基因烟草植株的PCR鉴定;B:1—6.转基因烟草植株的RT-PCR鉴定。

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.

图6 GUS基因在转基因pCAM-GmP34P烟草各组织中的表达

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

图7 GUS组织化学染色分析 WT.野生型烟草种子;GmP34P.转化pCAM-GmP34P表达载体的烟草种子;CaMV35S.转化pCAMBIA1301表达载体的烟草种子。

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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