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

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