【作者】 王志军；

【导师】 孙杰； 李艳军；

【作者基本信息】 石河子大学 ， 作物学， 2022， 博士

【摘要】 目的:棉花具有“无限生长”的特性,生产上常因不良气候或水肥管理不当导致棉花的营养生长和生殖生长不协调,造成弱苗迟发、徒长、贪青晩熟、吐絮不集中等,对高产稳产十分不利。缩节胺（Mepiquat chloride,DPC）可以协调棉花营养生长与生殖生长,促进养分适时运输至生殖器官,是实现棉花优质、高产、高效的有效措施之一。在众多植物生长调节剂中,DPC因其在棉花体内低残留、低毒、见效快和使用成本低等优势已经成为使用最广泛的棉田化控用药。生产实践证明,合理使用DPC具有明显的增产效应,西北内陆棉区增产15%左右,内地棉田一般增产8%～10%。本研究拟以DPC敏感棉花品系石大1068（SD1068）和DPC迟钝棉花品种新陆早74号（XLZ74）为材料,通过整合混池全基因组测序和转录组测序对DPC敏感性相关基因进行快速定位,对定位区段的候选基因进行功能注释,查找DPC敏感性相关基因及其他响应DPC化控的关键基因;对候选基因进行VIGS和基因编辑（CRISPR-Cas9）功能验证并解析候选基因的功能。研究结果将为DPC调控棉花生长的分子机制提供理论依据。方法:（1）前期研究中,根据田间农艺性状的表型,初步筛选出9个陆地棉材料,采用不同浓度DPC（40mg/L、80mg/L、120mg/L）喷施棉花,根据株高抑制程度,筛选最合适的处理浓度。（2）对陆地棉品种资源进行DPC敏感性田间鉴定,参考石治鹏（2015）划分标准,筛选陆地棉对DPC敏感和迟钝的品种。（3）对DPC敏感和迟钝品种开展DPC处理前后光合生理指标、内源激素含量、幼茎横切面细胞形态研究,明确DPC对上述指标的影响。（4）对DPC敏感和迟钝亲本DPC处理前后的主茎节间组织进行转录组测序（RNA-Seq）分析,筛选DPC响应相关基因。（5）利用混池全基因组测序对DPC敏感性相关位点进行快速定位,对定位区段的候选基因进行功能注释,结合转录组测序结果筛选DPC敏感相关基因,并采用实时荧光定量PCR技术（q RT-PCR）对其表达量进行检测。（6）对候选基因Gh TCP12（GH＿D12G1898）和Gh GA20ox1B（GH＿D09G0053）构建棉花基因沉默（VIGS）载体和基因编辑载体（CRISPR-Cas9）,检测沉默植株对DPC的敏感性变化,利用靶位点测序技术对基因编辑类型进行检测。结果和结论:（1）田间栽培试验中,9个陆地棉材料对DPC的敏感性差异较大,3个处理浓度对棉花株高均有抑制作用,且随着浓度升高,抑制程度逐渐加重。40mg/L DPC处理是最适合陆地棉敏感性鉴定,其次是80mg/L,DPC浓度为120mg/L时抑制程度较重,DPC敏感和迟钝材料的株高均被显著抑制;根据DPC对株高的抑制程度,对DPC敏感性进行等级划分,最终确定DPC迟钝材料为XLZ74,DPC敏感材料为SD1068。（2）与清水处理相比,DPC处理后,陆地棉叶绿素含量在所有处理中均显著升高（P<0.05）,SPAD值增幅达到7.11%-38.62%;DPC处理后,大多数处理的光合速率（P_n）,蒸腾速率（T_r）,气孔导度（G_s）,胞间CO₂浓度（C_i）不同程度升高。（3）转录组测序结果表明,与XLZ74相比,SD1068中赤霉素降解基因（GA2ox）在DPC处理3天和6天上调表达,而GA合成基因（GA20ox,GA3ox）富集到的数量较少,与激素检测结果GA4含量下降一致;大量的生长素负调节基因（AUX/IAA）在DPC处理6天上调表达,驱动生长素合成基因（ARF）的下调表达,生长素下游基因（GH3）也下调表达,激素检测结果也发现IAA含量降低;虽然CTK降解相关基因（CKX）在XLZ74和SD1068中均上调表达,SD1068中CTK合成基因（LOG）也上调表达,而XLZ74中CTK负调控因子（ARR）上调表达,激素检测结果中,SD1068中CTK含量显著增加,XLZ74增加不显著;SD1068中油菜素内脂合成相关基因下调表达,XLZ74中上调表达。此外,SD1068中大多数转录因子（TFs）的表达显著降低,相比之下,在XLZ74中发现较少下调的TFs,再次表明DPC对SD1068的抑制作用强于XLZ74。对XLZ74和SD1068经DPC处理前后的幼茎横切面做石蜡切片分析,DPC处理后2个材料的韧皮部和形成层细胞的直径,细胞面积显著降低,相同面积细胞数量显著增加。（4）亲本XLZ74×SD1068杂交组合中,DPC处理后,F₁、BC₁株高介于母本XLZ74（DPC迟钝）和父本SD1068（DPC敏感）之间,F₂群体株高分布呈连续变化,分布频率呈典型的正态分布,因此可以确定,DPC敏感性状属于数量遗传性状,可能有多个基因作用位点。利用转录组结合BSA混池测序定位技术,筛选到2个DPC敏感相关基因Gh TCP12和Gh GA20ox1B。Gh TCP12基因可调控节间伸长和激素合成,Gh GA20ox1B是编码赤霉素合成的关键限速酶,DPC处理后,Gh TCP12在XLZ74中显著上调表达,SD1068中呈显著下调表达,Gh GA20ox1B在XLZ74和SD1068中均上调表达,在XLZ74上调幅度更大。（5）对候选基因Gh TCP12和Gh GA20ox1B进行VIGS和基因编辑验证,结果发现,沉默植株中Gh TCP12和Gh GA20ox1B在叶片和茎中的表达量均显著下调,DPC处理后,株高抑制不显著,初步说明Gh TCP12和Gh GA20ox1B为DPC敏感基因。对Gh TCP12和Gh GA20ox1B遗传转化再生植株是否发生基因编辑进行PCR检测,检测到Cas9蛋白基因的植株为阳性株,本研究获得Gh GA20ox1B基因编辑阳性株5株,Gh TCP12基因编辑阳性株2株,通过对靶位点进行测序,检测到的基因编辑类型主要为碱基缺失和错配。更多还原

【Abstract】 Objective:Cotton has“indeterminate growth”habit.In cotton production,due to the poor climate and improper management of water and fertilizer,it will lead to uncoordinated vegetative and reproductive growth of cotton,it will also occur weak seedlings,excessive growth,over-green and late-maturing,vomiting not concentrated,very unfavorable high and stable yield of cotton.Therefore,the use of growth regulators to coordinate the vegetative and reproductive growth and ensure transfer nutrients to reproductive organs timely is an effective measure to ensure high quality,high yield and high efficiency of cotton.Among so many plant growth regulators,mepiquat chloride（DPC）has gradually become the most important chemical control drug in cotton because of its advantages of low toxicity,low residue,quick effect and low cost,practice of production.The practice of production has proved that the rational use of DPC has obvious yield increased.The yield increase in northwest inland reach to 15%,and that in inland generally increased by 8%～10%.In this study,DPC sensitive varieties（SD1068）and insensitive cotton varieties（XLZ74）were used as materials.Rapid localization of DPC-sensitive genes by integrated pooled whole genome sequencing and transcriptome sequencing,candidate genes were annotated in the localization region,and search for DPC-sensitive genes and other key genes responding to DPC.The functional of candidate genes were verified by VIGS and gene editing.The function and response to DPC of candidate gene was analysised.The results will provide a theoretical basis for the molecular mechanism of DPC regulating cotton growth.Methods:（1）According to previous study on phenotypic of agronomic traits on field,nine upland cotton materials were screened preliminarily.DPC were sprayed on cotton with different concentrations（40mg/L,80 mg/L and 120mg/L）,the most suitable concentration treatment was selected according to the inhibition of plant height.According to the inhibition degree of plant height,the most appropriate treatment concentration was selected.（2）The DPC sensitivity of upland cotton variety identification were carried out in field,and classification standard of upland cotton to DPC sensitive and insensitive reference to Shi ZP（2015）.（3）The photosynthetic physiological indexes,content of endogenous hormone,cell size of young stem before and after DPC treatment were studied to clarify the effects of DPC on those indexes.（4）Transcriptome sequencing（RNA-seq）analysis of the effects of DPC on the internodes of DPC sensitive and insensitive parents before and after DPC treatment,to screen DPC response related genes.（5）Bulk segregant analysis（BSA）was used to rapid locate the DPC sensitive related genes,functional annotation of candidate genes for locating regions.Combined with result of transcriptome sequencing,DPC-sensitive genes were screened and their expression levels were detected by q RT-PCR.（6）The candidate genes of Gh TCP12（GH＿D12G1898）and Gh GA20ox1B（GH＿D09G0053）were used to construct vector of Virus-induced gene silencing（VIGS）and gene editing（CRISPR-Cas9）.The objective genes expression of silencing plants were detected by q RT-PCR,and the gene editing type was detected by next-generation sequencing technologies.Results and conclusions:（1）In the field cultivation experiment,the sensitivity of nine upland cotton materials to DPC was quite different.Three concentrations had inhibitory effects on cotton plant height,and with the increase of concentration,the degree of inhibition gradually increased.40mg/L DPC was the most suitable for sensitivity identification of upland cotton,followed by 80mg/L DPC.When the DPC concentration was 120mg/L,the inhibition was severe,and the plant height of DPC sensitive and insensitive materials was significantly inhibited.According to the inhibition degree of DPC on plant height,the sensitivity of DPC was graded.Finally,the DPC insensitive material was XLZ74,and the DPC sensitive material was SD1068.（3）Compared with water treatment,the chlorophyll content of XLZ74 and SD1068 increased significantly after DPC treatment（P<0.05）,and the SPAD value increased by 7.11%～38.62%.After DPC treatment,the photosynthetic rate（P_n）,transpiration rate（T_r）,stomatal conductance（G_s）and intercellular CO₂ concentration（C_i）of most treatments was increased.（4）The results of transcriptome sequencing showed that,compared with XLZ74,the expression of gibberellin degradation gene（GA2ox）in SD1068 was up-regulated after 3 and 6 days by DPC treatment,while the number of GA synthesis genes（GA20ox,GA3ox）was less,which was consistent with the decrease of GA4 content by hormone detected.A large number of auxin negative regulator genes（AUX/IAA）were up-regulated after 6 days by DPC treatment.Due to the down-regulated expression of auxin synthesis gene（ARF）,and down-regulated expression of auxin downstream gene（GH3）,resulting in the content of IAA decreased.Although the CTK degradation-related gene（CKX）was up-regulated in both XLZ74 and SD1068,the CTK synthesis gene（LOG）in SD1068 was also up-regulated,and the CTK negative regulatory factor（ARR）in XLZ74 was up-regulated,so the CTK content in SD1068 was increased significantly,while in XLZ74 was not significantly increased.The down-regulated expression of genes related to brassinolide synthesis and up-regulated expression in XLZ74 led to lower BR content in SD1068.In addition,the expression of most TFs in SD1068 was significantly decreased.In contrast,less down-regulated TFs were found in XLZ74,indicating that DPC had a stronger inhibitory effect on SD1068than XLZ74.Paraffin sections of young stems of XLZ74 and SD1068 before and after DPC treatment were analyzed.The diameter and area of phloem and cambium cells of the two materials after DPC treatment were analyzed.（5）In the hybrid combination of XLZ74×SD1068,the plant heights of F₁ and BC₁ between the female parent XLZ74（DPC insensitive）and the male parent SD1068（DPC sensitive）.The plant height distribution of F₂ population showed continuous changes,and the distribution frequency showed a typical normal distribution.Therefore,it can be determined that the DPC sensitive traits were quantitative genetic traits,and there may have multiple gene action sites.Two DPC sensitive genes of Gh TCP12 and Gh GA20ox1B were screened by transcriptome analysis combined with BSA mixed pool sequencing Gh TCP12 gene can regulate internode elongation and hormone synthesis.Gh GA20ox1B encodes the key rate-limiting enzyme for gibberellin synthesis.After DPC treatment,Gh TCP12 was significantly up-regulated in XLZ74,significantly down-regulated in SD1068,and Gh GA20ox1B was up-regulated in both XLZ74 and SD1068,with a greater up-regulation in XLZ74.（6）The functional of candidate genes of Gh TCP12 and Gh GA20ox1B was verified by VIGS and gene editing technique.The results showed that the expression levels of Gh TCP12 and Gh GA20ox1B in leaves and stems in silencing plants were significantly down-regulated.After DPC treatment,plant height inhibition was not significant,which preliminarily indicated that Gh TCP12 and Gh GA20ox1B were DPC sensitive genes.PCR was performed to detect whether gene editing occurred in the regenerated plants of Gh TCP12 and Gh GA20ox1B genetic transformation,and have target sites of the Cas9 protein gene plants were sequenced.We obtained five gene editing positive strains of Gh GA20ox1B and two gene editing positive strains of Gh TCP12.It was found that the gene editing types were mainly base deletion and mismatch.更多还原