短花针茅StbCRY1和StbCRY2基因克隆与表达差异分析

doi:10.16258/j.cnki.1674-5906.2023.05.005

生态环境学报 ›› 2023, Vol. 32 ›› Issue (5): 866-877.DOI: 10.16258/j.cnki.1674-5906.2023.05.005

短花针茅StbCRY1和StbCRY2基因克隆与表达差异分析

赵鸿彬¹^,²^,³^,^*(), 白雪¹, 范宇凤¹, 张晓馥¹, 张涛¹, 李淑芬¹^,³

1.内蒙古农业大学生命科学学院，内蒙古呼和浩特 010018
2.内蒙古自治区生物制造重点实验室，内蒙古呼和浩特 010018
3.内蒙古高校麦类作物种质创新与利用重点实验室，内蒙古呼和浩特 010018

收稿日期:2022-12-13 出版日期:2023-05-18 发布日期:2023-08-09
通讯作者: *
作者简介:赵鸿彬（1978年生），女，副教授，研究方向为草原生态与牧草分子育种。E-mail: hbzhao04@163.com
基金资助:
国家自然科学基金项目(31760154);内蒙古自然科学基金项目(2021MS03094)

Cloning and Differential Expression Analysis of Stipa Breviflora StbCRY1 and StbCRY2 Genes

ZHAO Hongbin¹^,²^,³^,^*(), BAI Xue¹, FAN Yufeng¹, ZHANG Xiaofu¹, ZHANG Tao¹, LI Shufen¹^,³

1. College of Life Science, Inner Mongolia University, Hohhot 010010, P. R. China
2. Key Laboratory of Biological Manufacturing of Inner Mongolia Autonomous Region, Hohhot 010010, P. R. China
3. Key Lab of Germplasm Innovation and Utilization of Triticeae Crop at Universities of Inner Mongolia Autonomous Region, Hohhot 010010, P. R. China

Received:2022-12-13 Online:2023-05-18 Published:2023-08-09

摘要/Abstract

摘要：

短花针茅（Stipa breviflora）是荒漠草原的优势物种，兼具优秀的饲用价值和稳固水土的重要作用，其生殖生长受外界环境的影响较大。探究野外短花针茅生长发育过程中的CRY1、CRY2蛋白对不同环境条件的响应关系，可以为进一步研究蓝光受体隐花色素在不利环境条件下短花针茅生长发育中的调控机制提供有用的信息。以短花针茅转录组数据为基础，利用RACE技术得到生物钟相关基因StbCRY1和StbCRY2 ORF，其编码区分别为2695 bp和2176 bp。二者编码酸性蛋白质，皆是亲水蛋白质。StbCRY1蛋白具有PhrB结构域和Cry-C结构域，StbCRY2蛋白具有PhrB结构域。进化树分析表明短花针茅CRY1蛋白与野生二粒小麦（Triticum dicoccoides）和普通小麦（Triticum aestivum）CRY1进化关系较为相近，短花针茅CRY2蛋白与大麦（Hordeum innermongolicum）CRY2蛋白亲缘关系相近。利用生物信息学工具预测蛋白质二级结构，两种蛋白质出现α螺旋、无规线团概率为40%左右，出现β转角、延伸链的概率较小。共聚焦定位分析表明StbCRY1和StbCRY2主要定位于细胞核，少量定位于细胞质。同时利用实时荧光定量PCR技术对不同环境条件、不同开花时期短花针茅营养枝与生殖枝中StbCRY1和StbCRY2基因的表达差异进行分析发现，在增温施氮处理下，短花针茅生殖枝两种基因在开花期表达水平受增温和施氮处理的影响较大，增温施氮交互作用下影响尤为明显，开花中期表达量下调最为显著；营养枝叶片中StbCRY1和StbCRY2主要对增温处理有较明显的响应，不同开花时期表达量都与对照组表达量存在显著差异。该文可以为进一步探究气候变化情况下荒漠草原短花针茅生长发育的分子响应机制以及提供基础数据。

关键词: 短花针茅, StbCRY1, StbCRY2, 表达分析, 亚细胞定位, 基因克隆

Abstract:

Stipa breviflora is a dominant species in desert grassland, with excellent feeding value and important role in stabilizing soil and water. Its reproductive growth is greatly affected by the external environment. Exploring the response relationship of CRY1 and CRY2 proteins to different environmental conditions during the growth and development of Stipa breviflora in the field can provide useful information for further studying the regulation mechanism of blue light receptor cryptochrome in the growth and development of Stipa breviflora under adverse environmental conditions. Based on the transcriptome data of Stipa breviflora, the coding regions of the biological clock related genes StbCRY1 and StbCRY2 ORF obtained by using RACE technology were 2695 bp and 2176 bp, respectively. Both encode acidic proteins are hydrophilic proteins. StbCRY1 protein has a PhrB domain and a Cry-C domain, while StbCRY2 protein has a PhrB domain. Phylogenetic tree analysis showed that the CRY1 protein of Stipa breviflora is closely related to the CRY1 protein of wild wheat (Triticum dicoccoides) and common wheat (Triticum aestivum), while the CRY2 protein is closely related to the CRY2 protein of Hordeum innermongolicum. Using bioinformatics tools to predict their secondary structures, both proteins appeared α spiral; the probability of random coil was about 40%; the probabilities of β-turn and extending chains were relatively low. Through bioinformatics and confocal localization analysis, it was found that the StbCRY1 and StbCRY2 were mainly localized in the nucleus, with a small amount in the cytoplasm. At the same time, real-time fluorescence quantitative PCR technology was used to analyze the expression differences of StbCRY1 and StbCRY2 genes in the vegetative and reproductive branches of Stipa breviflora, that were collected under different environmental conditions and flowering stages. It was found that under warming and nitrogen application treatments, the expression levels of the two genes in the reproductive branches of Stipa breviflora during flowering stage were significantly affected by the treatments. And the flowering stage was particularly affected by the interaction of warming and nitrogen application. The downregulation of their expression level was most significant in the mid flowering stage; the expression levels of StbCRY1 and StbCRY2 in nutrient branches and leaves were significantly different from those in the control group at different flowering stages, as they mainly responded to the warming treatment. This study can provide basic data for further investigation of the molecular mechanism of how Stipa breviflora growth and development respond to the climate change in desert steppe.

Key words: Stipa breviflora, StbCRY1, StbCRY2, expression analysis, subcellular localization, gene cloning

中图分类号:

Q812.29
X173

赵鸿彬, 白雪, 范宇凤, 张晓馥, 张涛, 李淑芬. 短花针茅StbCRY1和StbCRY2基因克隆与表达差异分析[J]. 生态环境学报, 2023, 32(5): 866-877.

ZHAO Hongbin, BAI Xue, FAN Yufeng, ZHANG Xiaofu, ZHANG Tao, LI Shufen. Cloning and Differential Expression Analysis of Stipa Breviflora StbCRY1 and StbCRY2 Genes[J]. Ecology and Environment, 2023, 32(5): 866-877.

图/表 12

表1 扩增引物序列及用途

Table 1 The sequence and usages of the primers

序列名称	序列用途	序列
StbCRY1-F1	ORF cloning	CACTGCCCACGCACTCA
StbCRY1-R1	ORF cloning	GCAACAACCAAAGGCAAT
StbCRY2-F1	ORF cloning	GTTGTTCCATTCCGTCAC
StbCRY2-R1	ORF cloning	TGATGAGAGTGGAATACGGGA
5′ StbCRY1-GSP1	5′ cDNA RACE	GTTTCAGCCCTGCCTAAAGAG
5′ StbCRY1-GSP2	5′ cDNA RACE	CCTTCTTCCATACCGTTCTCCAT
5′ StbCRY2-GSP1	5′ cDNA RACE	CATCCCGCACAAGTGAAATAG
5′ StbCRY2-GSP1	5′ cDNA RACE	AGCGACTTCTTGAGCCACCAC
StbCRY1-GFP-F	Gene cloning	TTCATTTGGAGAA CACGGGGGAC
StbCRY1-GFP-R	Gene cloning	CAAGACCGGCAA CAGGATTCAATA
StbCRY2-GFP-F	Gene cloning	TTCATTTGGAGAA CACGGGGGAC
StbCRY2-GFP-R	Gene cloning	CAAGACCGGCAA CAGGATTCAATA
internal TLF Upstream	Gene expression analysis	CCCTCAGTGTG TGTTTGACC
internal TLF Downstream	Gene expression analysis	CTTGAGACCC TTCCTCTTGC
StbCRY1 Upstream	Gene expression analysis	TCATCAGCAGC GTAAGCATCT
StbCRY1 Downstream	Gene expression analysis	CGTCCACCAC CAGCAGTAT
StbCRY2 Upstream	Gene expression analysis	GCCAGTCCCAGG TATAGAGAATC
StbCRY2 Downstream	Gene expression analysis	AGGAGACCAC GAAGCACAA

图1 StbCRY1基因ORF及推导的氨基酸序列

Figure 1 The ORF and deduced protein sequence of StbCRY1

图2 StbCRY2基因ORF及推导的氨基酸序列

Figure 2 The ORF and deduced protein sequence of StbCRY2

表2 StbCRY1和StbCRY2基因编码蛋白的结构特征

Table 2 Structural characteristics of proteins encoded by StbCRY1 and StbCRY2 genes

一级结构特性	ORF长度/bp	推导氨基酸数	理论等电点 (PI)	分子量	不稳定系数 (II)	总平均亲水性	脂肪系数 (AI)	负电荷残基	正电荷残基
CRY1	2102	699	5.48	78829.36	51.84	-0.464	76.07	93	72
CRY2	1962	653	5.53	73429.95	45.5	-0.387	82.25	88	72

图3 StbCRY1和StbCRY2蛋白的空间构象预测

Figure 3 Prediction of the soatial conformation of StbCRY1 and StbCRY2 proteins

图4 StbCRY1、StbCRY2与其他物种CRY蛋白进化树

Figure 4 Phylogenetic tree of StbCRY with CRY1, StbCRY2 proteins from other plants species

图5 488 nm激发光下StbCRY1蛋白在烟草叶片中的表达情况

Figure 5 Expression of StbCRY1 protein in tobacco leaves under 488 nm excitation light

图6 488 nm激发光下StbCRY2蛋白在烟草叶片中的表达情况

Figure 6 Expression of StbCRY2 protein in tobacco leaves under 488 nm excitation light

图7 不同增温施氮条件下生殖枝StbCRY1和StbCRY2同一花期表达差异

Figure 7 Expression differences of StbCRY1 and StbCRY2 in reproductive branches at the same flower development stage under different temperature and nitrogen application conditions

图8 不同增温施氮条件下营养枝叶片StbCRY1和StbCRY2同一花期表达差异

Figure 8 Expression differences of StbCRY1 and StbCRY2 in vegetative branch leaves leaves at the same flower development stage under different temperature and nitrogen application conditions

图9 同一增温施氮条件下不同花发育时期生殖枝StbCRY1和StbCRY2表达差异

Figure 9 Expression differences of StbCRY1 and StbCRY2 in reproductive branches at different flower development stages under the same temperature and nitrogen application conditions

图10 同一增温施氮条件下不同花发育时期营养枝叶片StbCRY1和StbCRY2表达差异

Figure 10 Expression differences of StbCRY1 and StbCRY2 in vegetative branch leaves leaves at different flower development stages under the same temperature and nitrogen application conditions

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短花针茅StbCRY1和StbCRY2基因克隆与表达差异分析

Cloning and Differential Expression Analysis of Stipa Breviflora StbCRY1 and StbCRY2 Genes

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