中国农业科学Issue(12)：2306-2316,11.DOI:10.3864/j.issn.0578-1752.2015.12.003
棉花钠尿肽基因GhPNP1的耐旱功能分析
Drought-Enduring Functional Analysis of a Natriuretic Peptide GeneGhPNP1 in Cotton
摘要
Abstract
Objective] The objectives of this research are to analyze the structural features, patterns of expression and drought tolerance functions of natriuretic peptide geneGhPNP1 in cotton, and provide a theoretical basis for future deeper study of crop drought tolerance.[Method] The homolog protein sequences of plant natriuretic peptides in cotton were obtained by a BLASTP search inGossypium raimondiiprotein database using the query protein sequence ofVerticillium dahliae plant natriuretic peptides gene Ave1 acquired from plants through horizontal gene transfer. Homologous analysis and multiple alignments were performed with MEGA 5. Expasy online tools were used for protein sequence analysis. TheGhPNP1 was amplified inGossypium hirsutumAo3503 using the primers that were designed according to the nucleotide acid sequence of the homolog protein sequence. The isoelectric point, molecular weight, signal peptide and phylogenetic tree of the encoding protein were analyzed by related bioinformatics programs. The expression analysis ofGhPNP1 gene in different organs and by PEG simulating drought stress treatment was conducted by real-time PCR. The virus induced gene silence vector was constructed and theGhPNP1 silenced cotton plants were obtained. The cDNA sequences ofGhPNP1was add to CLCrV silencing vector to construct virus-induced gene silencing vector CLCrV: GhPNP1 of GhPNP1. At 2-leaf-stage, cotton plant was infected and leaf tissues were obtained from hormone treated plant. PEG simulating drought stress treatment was used to determine the drought tolerance ofGhPNP1silenced cotton plants and test the several physiological indexes related to stress tolerance, such as water loss rate, relative water content, MDA content, T-AOC level and electrolyte leakage inGhPNP1silenced cotton plants.[Result]GhPNP1 obtained inGossypium hirsutum Ao3503 has an ORF with 396 nucleotides and encodes a protein of 131 amino acids, its isoelectric point is 9.13, the predicted molecular weight is 14.6 kD and with a signal peptide of 15 amino acids. The protein contained a conserved natriuretic peptides domain and with the highest similarity ofGhPNP1 inTheobroma cacao. Quantitative real-time PCR (qRT-PCR) was used to examine the expression pattern of GhPNP1 in drought stress treatment.GhPNP1exhibited weakly organ different expressed pattern with a little stronger expressed in stem, and moderately expressed in cotton root and leaves. In addition, PEG simulating drought stress treatment induced the up-regulated expression ofGhPNP1 significantly in all the organs. The drought tolerance ofGhPNP1 silenced cotton plants decreased significantly. The MDA content, electrolyte leakage and water loss rate ofGhPNP1silenced cotton plants were significantly higher than un-silenced cotton plants, wherever the T-AOC level and relative water content of silenced cotton plants were significantly lower than the un-silenced cotton plants.[Conclusion]A plant natriuretic peptides gene (GhPNP1) was cloned from cotton, which is induced up-regulated by PEG simulating drought stress treatment andGhPNP1 silenced cotton plants significantly decreased drought tolerance. These imply thatGhPNP1 may affect the drought tolerance of cotton through a cGMP dependent regulation pathway.GhPNP1 may play a positive role in drought tolerance of cotton.关键词
陆地棉/病毒诱导的基因沉默/干旱胁迫/功能分析Key words
upland cotton/virus induced gene silence/drought stress/function analysis引用本文复制引用
刘小双,刘廷利,袁洪波,张保龙,王荣富..棉花钠尿肽基因GhPNP1的耐旱功能分析[J].中国农业科学,2015,(12):2306-2316,11.基金项目
国家自然科学基金青年科学基金（31401694）、江苏省自然科学基金青年基金（BK20140743）、江苏省农业科技自主创新资金 （13）
