Identifying For Thermotolerance Of Cotton Germplasm Resources And Study The Heat Stress Effect

Periodic heat damage happened frequently in cotton planting areas of China, especially in Yangtze Rivervalleys. Screening of heat resistent germplasm and breeding of new heat resistant varieties are theimportant ways to guarantee the high-yield production of cotton. For this purpose, the establishment ofefficient indoor evaluation system and research of the key genes and elements related with heat resistantare significant for developing the heat resistant cotton varieties. In this study, the characters of heatresistant were understood primarily after200cotton varieties which planted in the naturalhigh-temperature area (Turpan Basin in Xinjiang province, China) were assessed by assaying the relatedheat tolerance traits. Based on the treatment the above germplasm with clear understand of thecharacters of heat resistance with different temperature during different seedling period, and byinvestigating of chlorophyll content, relative conductivity, SOD、POD、CAT、MDA、APX、ASA、GSH and GR, the rapid cotton heat resistant evaluation system was constructed. On molecular level, thefull-length sequences and expression level of HSP genes were obtained and surveyed by RACE,Genome Walking and Quantitive RT-PCR technology. The analysis of small RNA in different varietiesand treatments were performed by high-throughput sequencing technology. The main results were asfollowing:(1).200different cotton germplasm planted in Xinjiang were investigated for boll dropping rate,infertility seed rate, degree of leaf wilting, pollen morphology and pollen viability,29varieties withdifferent heat resistance were screened.(2).High heat resistant varieties-Nandanbadidahua and heat sensitive varieties-Zaoshuchangrong7were treated at30℃、35℃、40℃、45℃and48℃for8hours in different growing stages.40℃for8hours in three-leaves stage was determined as the optimized treatment for indoor identification of heatresistant in cotton.8physiological and biochemical index (OD、POD、CAT、MDA、APX、ASA、GSH and GR) could be used as identification index, but only former5need to be considered for rapididentification in seedling stage.(3) To illuminate the molecular mechanism for heat tolerance cotton, the heat shock protein(HSP)were analyzed thoroughly in this study.Firstly, the whole cDNA, DNA and5’ regulator seaueces ofGhHSP18.5, GhHSP26and cytoplasm GhHSP70(cGhHSP70) in cotton were gained.Then, theexpressions of heat shock protein were analyzed by using Qrt-pcr. It was found that the tolerantmaterials―Nandanbadidahua‖had higher expression levels of heat shock protein than that of heatsensitive materials―Zaoshuchangrong7‖. Moreover, the upstream regulatory sequence of GhHSP18.5and GhHSP26contained heat shock regulatory element (HSE) in Nandanbadidahua with heat resistance,but there had not HSE in that of GhHSP26in zao shuchangrong7with heat sensitive. (4) By race technique, it was found cGhHSP70have two isoenzyme cGhHSP70-1andcGhHSP70-2in zao shuchangrong7with heat sensitive, and an extra sequence of238bp in cGhHSP70-1was an intron sequence with a termination code. Meanwhile, the same expression of splicing factorRSp41, RSp35, RSp45was consistent with that of cGhHSP70-1. These resulted in null cGhHSP70-1transcript accumulation and cGhHSP70function for heat tolerance lossed in zao shuchangrong7.According to the results of high throughput sequencing sRNAs, miRNA1859with the target encodingRSp41was higher expression in―Nandanbadidahua‖than that in―zaoshuchangrong7‖. There are sevenendogenous miRNA targets with RSp family. It implied the cotton endogenous miRNA inhibit theexpression of RSp family, and reduce the accumulation of null transcript at heat shock.(5) By high-throughput sequencing technology,35known conserved miRNAs and172kinds ofnew miRNA (nmiRNA) were acquired in the different cotton germplasm with different heat resistant.Some new miRNAs such as nmiRNA31, nmiRNA32, nmiRNA39, nmiRNA42, nmiRNA43, nmiRNA45,nmiRNA46, nmiRNA58, nmiRNA59, nmiRNA67, nmiRNA68and nmiRNA94only expressed in―Zaoshuchangrong7‖, and nmiRNA52, nmiRNA52, nmiRNA5261, nmiRNA62, nmiRNA63, nmiRNA64,nmiRNA65and nmiRNA66only were be observed in‖Nandanbadidahua‖. It suggests these newmiRNAs can be used as identified markers or probe for cotton thermotolerance.(6) The rRNA content in zaoshuchangrong7was higher than that in nandanbadidahua under hightemperature stress, we speculated that the degradation of rRNA in nandanbadidahua concerned withresponse of heat stress in cotton. Whatever before or after high temperature treatment, nandanbadidahuapossess more sRNA than zaoshuchangrong7, less siRNA. However, as treatment time went on. Thequantity of sRNA in both of the2varieties decreased, siRNA increased. Under heat stress, the first19ntU bias miRNA presented in library of high resitant., the first18nt or19nt C bias miRNA inzaoshuchangrong7, this result indicated that the type and quantity of miRNA were different betweenhigh resistant varieties and sensitive varieties.(7) Using miRNAs sequencing and their target gene expression analysis, we foundGh-miRNA2919targeted on heat shock protein70, Gh-miRNA2651targeted on MYB1, Gh-miRNA946on HDAC(histone deacetylase), Gh-miRNA444targeted on different rRNA, and other endogenousmiRNAs may involved in regulation of cotton heat response.