青杄<i>PwUSP</i>1基因特征及对干旱和盐胁迫的响应

刘峻玲; 梁珂豪; 苗雅慧; 胡安妮; 孙永江; 张凌云

doi:10.12171/j.1000-1522.20200063

摘要:

目的 USPs蛋白（universal stress proteins）是一类胁迫相关类蛋白，被广泛报道参与了植物应对非生物胁迫的过程。本研究通过对青杄中PwUSP1基因进行功能分析及验证，探索PwUSP1在植物应对盐和干旱胁迫时的作用，从而为未来通过转基因工程提高青杄对非生物胁迫的耐受性提供候选基因。

方法通过瞬时转化烟草叶片实验揭示PwUSP1在细胞中的定位；利用酵母双杂实验鉴定PwUSP1自身能否形成同源二聚体；通过农杆菌侵染法转化野生型拟南芥（WT），获得纯合的PwUSP1过表达株系。通过测定干旱和盐胁迫下过表达株系（L1、L7）及野生型（WT）和空载体（VC）株系的存活率、失水率，来分析比较不同株系对于干旱和盐胁迫的耐受能力；通过二氨基联苯胺（DAB）和氯化硝基四氮锉蓝（NBT）染色，测定超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）以及丙二醛（MDA）的含量，研究PwUSP1发挥作用的生理机制。

结果烟草亚细胞定位实验表明，PwUSP1定位于细胞核、细胞质和细胞膜中。酵母双杂结果显示PwUSP1蛋白自身能够形成同源二聚体。利用qRT-PCR检测转基因拟南芥，成功获得两个稳定纯合的株系（L1、L7）进行进一步分析。在盐和干旱胁迫下，相对于WT和VC，过表达PwUSP1能够显著提高植物对盐和干旱的耐受能力，表现出更高的存活率和更低的失水率，且显著降低了植株中过氧化氢、超氧阴离子的累积，提高了SOD、POD和CAT活性，抑制了MDA的积累。

结论青杄PwUSP1定位于细胞核、细胞质和细胞膜中且自身能够形成同源二聚体，在干旱和盐胁迫条件下，PwUSP1通过增强植物的ROS清除能力及抑制膜脂氧化损伤来提高植物对非生物胁迫的耐受性。

Abstract:

Objective Universal stress proteins (USP) are stress-related genes which are widely reported to participate in the process of abiotic-stress response of plants. By identifying and validating the function of PwUSP1, we revealed the role of PwUSP1 in plants under drought and salt stress, thereby providing candidate genes for improving the tolerance to abiotic stress through genetic engineering in Picea wilsonii.

Method Transient transformation of tobacco leaves was used to reveal the location of PwUSP1 in cells. Yeast two-hybrid experiment was used to determine whether PwUSP1 could form homodimers by itself. The Arabidopsis Col-0 (WT) was transferred by floral dip method to obtain homozygous PwUSP1 overexpression lines. The survival rates and water loss rates of PwUSP1 overexpression plants, wild type (WT) and empty vector (VC) were measured to analyze and compare the tolerance of different lines when plants were subjected to drought and salinity. DAB and NBT staining, the activities of SOD, POD, CAT and the content of MDA were determined to explore the potential physiological mechanism of PwUSP1 acting.

Result Subcellular localization results showed that PwUSP1 was located in the nucleus, cytoplasm and cell membrane. Besides, the yeast two hybrid experiment showed that PwUSP1 itself can form homodimers. qRT-PCR was used to detect transgenic Arabidopsis, and the results showed that two independent homozygous lines with stable overexpression (L1, L7) were successfully obtained for further analysis. Under drought or salt stress, compared with WT and VC, PwUSP1 overexpression lines significantly improved the drought or salt tolerance of plants, and showed higher survival rates and lower water loss rates. Furthermore, overexpression of PwUSP1 obviously reduced the content of H₂O₂ and O₂ ⁻, and simultaneously promoted the activities of antioxidant enzymes and inhibited the accumulation of MDA.

Conclusion PwUSP1 is located in the nucleus, cytoplasm and cell membrane, and can form homodimers by itself. Under drought or salt stress, PwUSP1 improves the tolerance to abiotic stress of plants by enhancing ROS scavenging ability and inhibiting membrane lipid peroxidation.

青杄PwUSP1基因特征及对干旱和盐胁迫的响应

Characteristics of PwUSP1 in Picea wilsonii and its response to drought and salt stress