荸荠淀粉分支酶<i>CwSBEII</i>基因的克隆及其表达分析

赵若男; 陈振林; 玉万国; 许薇; 刘英健; 宋慕波

doi:10.19303/j.issn.1008-0384.2022.012.005

荸荠淀粉分支酶CwSBEII基因的克隆及其表达分析

doi: 10.19303/j.issn.1008-0384.2022.012.005

赵若男^{1, 2,},
陈振林²,
玉万国¹,
许薇²,
刘英健²,
宋慕波^{1, 2, ,}

1.
广西科技大学生物与化学工程学院，广西　柳州　545616
2.
贺州学院食品科学与工程技术研究院，广西　贺州　542899

基金项目: 国家重点研发计划项目（2018YFD0901003）；国家自然科学基金项目（31801607）；广西壮族自治区自然科学基金项目（2020GXNSFAA259087、2017GXNSFAA198082）；贺州学院博士科研启动基金（HZUBS202106）

详细信息

作者简介:
赵若男（1997−），女，硕士研究生，研究方向：果蔬加工与保鲜（E-mail：zhao18278289315@163.com）

通讯作者:
宋慕波（1986−），男，博士，副研究员，研究方向：果蔬加工与保鲜（E-mail：songmubo1@163.com）

中图分类号: S 645.3
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- 文章访问数: 315
- HTML全文浏览量: 155
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- 被引次数: 0
出版历程
- 收稿日期: 2022-07-07
- 修回日期: 2022-10-18
- 网络出版日期: 2022-12-28
- 刊出日期: 2022-03-28

Cloning and Expression of SBEII in Eleocharis tuberosa

ZHAO Ruonan^{1, 2
,},
CHEN Zhenlin²,
YU Wanguo¹,
XU Wei²,
LIU Yingjian²,
SONG Mubo^{1, 2
, ,}

1.
College of Biological and Chemical Engineering, Guangxi University of Science and Technology, Liuzhou, Guangxi　545616, China
2.
Institute of Food Science and Engineering Technology, Hezhou University, Hezhou, Guangxi　542899, China

摘要

摘要: 目的克隆荸荠（Eleocharis tuberosa）淀粉分支酶CwSBEII基因，分析该基因的蛋白功能特性及其在荸荠不同组织以及不同膨大时期的表达模式，为进一步深入研究CwSBEII基因的功能提供理论参考。方法采用RT-PCR的方法克隆得到CwSBEII基因的cDNA序列，通过生物信息学分析其蛋白功能特性，利用荧光定量PCR法检测该基因在荸荠球茎中的时空表达特征。结果成功克隆得到CwSBEII基因，该基因的开放阅读框（ORF）长度为2547 bp，编码848个氨基酸。CwSBEII蛋白分子量为96100.52 Da；理论等电点（pI）值为5.24，不稳定指数为41.07，GRAVY为−0.473，属于不稳定亲水蛋白。系统进化分析表明，荸荠与禾本科的小麦（Triticum aestivum）、水稻（Oryza sativa）、玉米（Zea mays）的SBEII蛋白聚为一支，亲缘关系最近。荧光定量PCR结果表明，CwSBEII基因在根、叶片、荸荠皮以及荸荠肉中均有表达，但在叶片中的表达量最高。在荸荠球茎膨大初期CwSBEII基因表达出现明显上调。结论 CwSBEII基因在荸荠中特异性表达，在叶片中的表达量最高，膨大初期表达明显上调，推测该基因在荸荠淀粉合成中发挥着重要作用。
- 荸荠 /
- CwSBEII /
- 克隆 /
- 基因表达
Abstract: Objective CwSBEII in Eleocharis tuberosa was cloned to understand the functions and expressions of the gene in various tissues and corms at different expansion stages. Methods The cDNA sequence of CwSBEII of Chinese water chestnut was cloned by RT-PCR, functions deciphered by bioinformatics, and spatiotemporal expressions in tissues and corms determined by fluorescence quantitative PCR. Results CwSBEII had an ORF length of 2 547 bp encoding 848 amino acids. The molecular weight of this unstable hydrophilic protein was 96 100.52 Da with a theoretical pI of 5.24, an instability index of 41.07, and a GRAVY reading at −0.473. It was closely related to and clustered with the SBEIIs of wheat (Triticum aestivum), rice (Oryza sativa), and maize (Zea Mays) of the grass family. Although found in the roots, leaves, peels as well as corm, CwSBEII was most highly expressed in the leaves and significantly upregulated in the early stage of corm expansion of Chinese water chestnut. Conclusion CwSBEII was specifically expressed in various tissues of Chinese water chestnut and most highly in the leaves. It was significantly upregulated during the early stage of corm expansion and might play an important role in the starch synthesis of E. tuberosa.
- Eleocharis tuberosa /
- CwSBEII /
- cloning /
- gene expression

HTML全文

图 1 CwSBEII基因PCR扩增产物

M：DL5000 DNA Marker;1～2:目的基因片段。

Figure 1. Electrophoresis of PCR amplification products of CwSBEII

M: DL5000 DNA marker; 1–2: target gene fragment.

下载: 全尺寸图片幻灯片

图 2 CwSBEII蛋白疏水/亲水性预测

Figure 2. Predicted hydrophobicity and hydrophilicity of CwSBEII protein

下载: 全尺寸图片幻灯片

图 3 CwSBEII蛋白保守结构域分析

Figure 3. Conserved structural domain of CwSBEII protein

下载: 全尺寸图片幻灯片

图 4 CwSBEII蛋白二级结构预测

蓝色（h）：α-螺旋；红色（e）：延伸链；橙色（c）：无规则卷曲；绿色（t）：β-折叠。

Figure 4. Predicted secondary structure of CwSBEII protein

Blue (h): α-helix; red (e): extended chain; orange (c): random coils; green (t): β-sheet.

下载: 全尺寸图片幻灯片

图 5 CwSBEII蛋白质的三级结构预测

Figure 5. Predicted tertiary structure of CwSBEII protein

下载: 全尺寸图片幻灯片

图 6 CwSBEII所编码的蛋白质与其他植物蛋白的系统进化树

Figure 6. Phylogenetic tree of proteins encoded by CwSBEII and other plant proteins

下载: 全尺寸图片幻灯片

图 7 CwSBEII所编码的蛋白质与不同植物SBEII氨基酸序列的多重比对

#：高度保守的活性位点。

Figure 7. Multiplex comparison on amino acid sequences of proteins encoded by CwSBEII and plant SBEIIs

#: Highly conserved active site.

下载: 全尺寸图片幻灯片

图 8 CwSBEII基因在荸荠不同组织中的相对表达

图中不同小写字母表示5%水平差异显著。图9同。

Figure 8. Expressions of CwSBEII in tissues of Chinese water chestnut

Data with different lowercase letters represent significant difference at 5% level. Same for Fig.9.

下载: 全尺寸图片幻灯片

图 9 CwSBEII基因在荸荠膨大时期中的表达变化

S1～S4分别表示球茎最宽处直径约为10 、20 、35和50 mm。

Figure 9. Expressions of CwSBEII during expansion stages of Chinese water chestnut corm

S1-S4: diameters of widest part of corms being approximately 10, 20, 35, and 50 mm, respectively.

下载: 全尺寸图片幻灯片

表 1 荸荠SBEII基因克隆和表达所用引物

Table 1. Primer used for cloning and expression analysis of SBEII in Chinese water chestnut

引物名称 Primer name	引物序列（5′-3′） Sequence(5′-3′)
CwSBEII (F)	ATGACGTTCGCTCTATCGGGATCGG
CwSBEII (R)	TTACTCCTCACAGAGAGCATAGACA
Q-PCR CwSBEII (F)	CCTCCTGAAGAAGAAAAGTACGTC
Q-PCR CwSBEII (R)	AGCTAGCATAGTAAGAGTGCTCCTG
18S RNA (F)	ATGATTAAGAGGGACAGTCGGGGGC
18S RNA (R)	CTAGGACGGTATCTGATCGTCTTCG

下载: 导出CSV

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