Nawaz Gul, Han Yue, Usman Babar, Liu Fang, Qin Baoxiang, Li Rongbai
College of Agriculture, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004 China.
3 Biotech. 2019 Jul;9(7):254. doi: 10.1007/s13205-019-1787-4. Epub 2019 Jun 7.
Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of in cold stress and 26 mutant plants were obtained in T generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T generation. The conserved amino acid sequence was changed and the expression level of was reduced in mutant plants. The mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (, , and ) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.
富含脯氨酸的蛋白（PRPs）在植物生长和胁迫响应中发挥多种生理和生化作用。在本研究中，我们报道了[基因名称]的敲除导致水稻对冷敏感。利用CRISPR/Cas9技术构建突变体植株，以研究[基因名称]在冷胁迫中的作用，在T代获得了26株突变体植株，突变率为85%，其中双等位基因占15%，纯合子占53.3%，杂合子占16.7%，T代有16个无T-DNA的株系。突变体植株中保守氨基酸序列发生改变，[基因名称]的表达水平降低。[基因名称]突变体植株对冷胁迫表现出更高的敏感性，与野生型（WT）相比存活率较低，根生物量减少，大片段缺失的纯合突变株系对低温更敏感。在低温胁迫下，突变株系相对于WT积累的抗氧化酶活性较低，脯氨酸、叶绿素、脱落酸（ABA）和抗坏血酸（AsA）含量也较低。对外源水杨酸（SA）处理的叶片和根中的抗氧化酶变化进行了检测，结果表明，冷胁迫下超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性增加，而未处理的幼苗中酶抗氧化活性较低，这表明外源SA预处理可以缓解水稻的低温胁迫。此外，与WT相比，突变株系中编码抗氧化酶活性的三个基因（[基因名称1]、[基因名称2]和[基因名称3]）的表达显著下调。这些结果表明，[基因名称]通过调节抗氧化剂和维持信号通路间的相互作用来增强耐寒性。因此，[基因名称]基因可用于提高水稻的耐寒性，CRISPR/Cas9技术有助于通过分析产生的敲除突变体的表型来研究基因的功能。
