额种子蛋白体靶向表达口服重组胰岛素原载体构建及转化水稻

doi:10.3969/j.issn.1001G7216.2015.05.005

摘要/Abstract

摘要：

应用水稻种子生物反应器开发口服重组胰岛素原具有重要应用前景。通过分子设计保证重组胰岛素原在人体肠道内的自主加工成熟,根据水稻密码子偏爱性人工合成了霍乱毒素β亚基和人胰岛素原的融合基因(cholera toxin B subunit fused with human proinsulin,CTBIN),并在C末端添加内质网滞留信号KDEL。通过PCR技术从粳稻品种日本晴全基因组中克隆谷蛋白启动子及其信号肽序列pGluB1sig(GluB1 promoter and its signal peptide)用于驱动融合基因CTBIN的表达,插入载体pCAMBIA1302,构建了水稻种子蛋白体靶向表达口服重组胰岛素原的载体pCAMBIA1302-pGluB1sig-CTBIN-Nos。采用农杆菌介导法转化日本晴,获得了46株转基因水稻植株,Western杂交检测到CTB-人胰岛素原融合蛋白在水稻种子中表达。

关键词: 口服重组胰岛素原, 蛋白体靶向表达, 载体构建, 转化, 水稻

Abstract:

Developent of oral recombinant proinsulin using rice seeds as bioreactor shows good application future. A fusion gene of the cholera toxin B subunit and human proinsulin (CTBIN) with an endoplasmic reticulum retention signal (KDEL) at C-terminus was designed for its self-maturation process in human gut and synthesized according to the optimized code usage bias of rice. The synthetic CTBIN was introduced to the vector pCAMBIA1302 and expressed under the 2.4 kb promoter sequence of rice glutelin GluB1 with its signal peptide (pGluB1sig), which was cloned by PCR from the japonica rice Nipponbare genome. Thus the rice seed protein body targeted expression vector pCAMBIA1302-pGluB1sig-CTBIN-Nos for oral delivery of recombinant proinsulin was constructed and then transformed into Nipponbare via Agrobacterium-mediated method. Totally 46 transgenic rice plants were obtained and the expression of the fusion protein CTB::Human proinsulin in rice seeds was detected by Western-blot.

Key words: oral recombinant proinsulin, protein body targeted expression, vector construction, transformaiton, rice

中图分类号:

唐湧洲, 俞越, 赵艳. 额种子蛋白体靶向表达口服重组胰岛素原载体构建及转化水稻[J]. 中国水稻科学, 2015, 29(5): 481-489.

Yong-zhou TANG, Yue YU, Yan ZHAO. Construction of Seed Protein Body Targeted Expression Vector and Transformation Rice for Oral Recombinant Proinsulin[J]. Chinese Journal OF Rice Science, 2015, 29(5): 481-489.

图/表 8

图1 口服重组胰岛素原融合基因CTBIN分子结构

Fig. 1. Structure of oral recombinant proinsulin fusion gene CTBIN.

图2 启动子及信号肽pGluB1sig 的PCR克隆(A)和中间载体的酶切鉴定(B)

Fig. 2. PCR cloning of GluB1 promoter with its signal peptide (A) and identification of the intermediate vector by restriction enzymes digestion (B).

图3 重组质粒pCAMBIA2301-pGluB1sig-CTBIN-Nos(A)和pCAMBIA1302-pGluB1sig-CTBIN-Nos(B)的酶切鉴定

Fig. 3. Identification of recombinant vectors pCAMBIA2301-pGluB1sig-CTBIN-Nos (A) and pCAMBIA1302-pGluB1sig-CTBIN-Nos (B) by restriction enzyme digestion.

图4 质粒pCAMBIA1302-pGluB1sig-CTBIN-Nos融合基因CTBIN启动子及编码区测序结果分析

Fig. 4. Sequencing result analysis of the promoter and coding region of fusion gene CTBIN in plasmid pCAMBIA1302-pGluB1sig-CTBIN-Nos.

图5 融合基因CTBIN转化水稻愈伤组织及植株再生

Fig. 5. Transformation of rice calli with fusion gene CTBIN and plantlets regeneration.

图6 部分T0代水稻转基因植株PCR检测结果

Fig. 6. PCR analysis of some T0 transgenic rice plants.

表1 农杆菌介导融合基因CTBIN转化水稻效率

Table 1 The transformation frequency of rice with fusion gene CTBIN via Agrobacterium transformation.

处理愈伤数 Treated callus number	抗性愈伤数 Resistant callus number	绿苗总数 Green plant number	绿苗分化率 Green plant differential rate /%	CTBIN基因 PCR阳性绿苗数 CTBIN PCR positive transgenic plant number	hpt基因 PCR阳性绿苗数 hpt PCR positive transgenic plant number	转化率 Frequency of transformation /%
102	22	48	218.2	46	48	13.7

图7 转基因水稻种子融合基因CTBIN表达的Western杂交检测

Fig.7. Western blot detection of fusion gene CTBIN expression in transgenic rice seed.

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