摘要
在我国棉花种植区域,特别是长江流域棉区,周期性的热害经常发生,筛选耐热种质,选育耐热新品种是解决热害,保证棉花高产的根本措施。而建立快速高效的室内高温鉴定体系,深入研究高温调控机理,明确耐高温有关的关键基因或作用元件,对培育耐高温品种具有重要意义。为此,本研究通过新疆吐鲁番自然高温条件下的田间耐热性鉴定,初步明确部分种质的抗感高温特性,利用这些材料在不同苗龄期在室内进行不同温度处理,并测定叶绿素、相对电导率、SOD、POD、CAT、MDA、APX、ASA、GSH和GR等十个生理生化指标,进而建立棉花室内耐热性快速鉴定系统。同时利用Race、染色体步移和普通PCR技术,获得棉花HSP18.5、HSP26、HSP70等热激蛋白的cDNA全长序列和DNA序列。然后通过荧光定量技术检测不同热激蛋白在不同耐热性材料中的表达水平,分析热激蛋白与棉花耐热性的关系。通过高通量测序技术和网络资源,分析不同耐热性材料在不同高温处理下,sRNAs种类和表达差异,预测棉花miRNAs在棉花耐热性中的功能。结果如下:
(1)在新疆,调查了200份不同耐热性种质的脱落率、不孕籽率、叶片萎蔫程度、花粉形态、花粉活力和花粉不同温度离体培养的萌发率等指标。初步筛选出29份不同耐热性棉花种质资源,用于室内耐热性鉴定。
(2)对耐高温材料常抗棉、南丹巴地大花以及高温敏感材料岱字棉55和早熟长绒7号于室内在子叶期、一叶一心期、二叶一心期、三叶一心期、四叶一心期和五叶一心期等不同苗龄期,分别进行30℃、35℃、40℃、45℃和48℃温度处理8h后,测定各项生理生化指标,初步建立了棉花耐热性室内鉴定体系:三叶期高温处理用于棉花耐热性鉴定的最佳叶龄期,最合适的鉴定温度是40℃处理8h,适合鉴定指标有相对电导率、MDA、APX、POD、SOD、CAT、GR、ASA等八项,如果是苗期快速鉴定,可以只考虑前五个鉴定指标。
(3)为揭示棉花高温耐性的分子机制,本研究对棉花热激蛋白(Heat Shock Protein, HSP)进行深入分析,首先获得了棉花HSP18.5、HSP26和cHSP70热激蛋白基因的cDNA序列、DNA序列和上游调控区。然后用荧光定量PCR分析热激蛋白的表达量,发现耐高温材料南丹巴地大花的热激蛋白基因表达量要高于高温敏感材料的表达量。进一步分析发现,耐高温材料南丹巴地大花的小热激蛋白HSP18.5和HSP26的上游调控序列中都含有HSE调控元件,但是在高温敏感材料早熟长绒7号HSP26的启动子区中没有热激调控元件(HSE)。
(4)通过Race,在高温敏感材料早熟长绒7号中获得到两个长度不同的cGhHSP70-1和cGhHSP70-2,通过分析发现,序列cGhHSP70-1中多出一段长度为238bp并包含终止密码子的内含子序列,同时剪接因子RSp41、RSp35、RSp45的表达量与cGhHSP70-1一致,造成了cGhHSP70-1等错误转录本的积累,从而破坏了早熟长绒7号中cGhHSP70的功能,进而影响该材料的耐高温性。根据sRNAs高通量测序的结果,发现miRNA1859以RSp41为靶基因,南丹巴地大花的Gh-miRNA1859表达量高于早熟长绒7号。棉花中还有7个内源miRNA与RSp相关。因此推测,棉花内源miRNA能够抑制RSp家族的表达,降低错误转录本的积累。
(5)通过高通量测序技术,在不同耐热性材料中获得已知35种保守的已知miRNA和172种新miRNA。其中新预测nmiRNA31、nmiRNA32、nmiRNA39、nmiRNA42、nmiRNA43、nmiRNA45、nmiRNA46、nmiRNA58、nmiRNA59、nmiRNA67、nmiRNA68和nmiRNA94只在早熟长绒7号中表达。而nmiRNA52、nmiRNA52、nmiRNA61、nmiRNA62、nmiRNA63、nmiRNA64、nmiRNA65和nmiRNA66只在南丹巴地大花中表达。因此推测这些新nmiRNAs可以用于鉴定棉花耐热性的标记或者探针。
(6)高温胁迫下,早熟长绒7号rRNA相对含量要高于南丹巴地大花,推测rRNA的降解与棉花热应激反应相关。无论是高温处理前,还是处理后,抗高温材料南丹巴地大花较高温敏感材料早熟长绒7号具有较多种类和数量的sRNA,较少种类和数量的siRNA;但随着高温处理时间的延长,抗高温和高温敏感材料的sRNA种类和数量都在逐渐减少,而siRNA种类和数量都在逐渐增加。在高温应激情况下,抗高温材料sRNA库可能出现首位碱基偏好U的19nt miRNA,高温敏感材料可能出现首位碱基偏好C的18nt或19nt miRNA。这说明不同类型的sRNA种类和数量在抗高温和高温敏感材料间是有差别的。
(7)miRNAs测序分析和靶基因荧光定量分析,以热激蛋白70为靶基因的Gh-miRNA2919、以MYB1为靶基因的Gh-miRNA2651、以组蛋白去乙酰化酶为靶基因的Gh-miRNA946,以不同类型rRNA基因为靶基因的Gh-miRNA444等内源miRNA参与调控了棉花的耐热性。
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.
引文
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