【作者】 刘敏；

【导师】 陶书田；

【作者基本信息】 南京农业大学 ， 农业硕士（专业学位）， 2020， 硕士

【摘要】 本试验基于前人的相关研究,将‘砀山酥梨’愈伤组织培养基配方做了进一步优化,对‘砀山酥梨’果肉进行愈伤组织的诱导,并以‘砀山酥梨’愈伤组织为主要材料,对愈伤组织进行不同浓度的钙处理,结合木质素含量、钙含量、表型等生理指标来筛选出抑制木质素形成的适宜钙浓度,以期对田间喷施钙肥抑制石细胞提供理论指导。筛选出钙处理后生理差异较大的愈伤组织处理组送转录组测序,对测序结果进行分析,以期获得钙调控木质素合成途径中可能参与的钙结合蛋白相关基因,进一步探究钙影响木质素形成的调控网络。基于转录组数据挑选候选基因家族PbCMLs,并进行其基因家族分析,为进一步研究PbrCML基因在梨发育过程中的功能及是否对木质素合成起调控作用提供了资源。结合实时荧光定量PCR技术,克隆基于转录组数据所获得的关键基因,通过梨幼果瞬时侵染、亚细胞定位技术,对PbrCML关键基因进行功能验证,为揭示钙调控木质素合成途径提供分子基础。主要结果如下:1、对砀山酥梨愈伤组织培养基配方做了进一步优化,即:MS 4.74 g/L+1.5 mg/L 2,4-D+0.5 mg/L NAA+0.2 mg/L TDZ+7 g/L 琼脂+30 g/L 蔗糖+10 g/L 山梨醇+0.1 mg/L VC,成功诱导‘砀山酥梨’愈伤组织,建立了较为完善的愈伤组织培养技术体系。试验基于前人田间氯化钙喷施实验之上,通过不同浓度的硝酸钙处理砀山酥梨果实的愈伤组织,以离体验证钙对愈伤组织木质素含量的影响,进一步探究抑制梨果实石细胞形成的适宜浓度。结果表明,硝酸钙处理对梨果实愈伤组织具有一定的影响,经0.1%浓度硝酸钙处理后的愈伤组织生长速度较快,生长状态佳,表面湿润,且随着处理时间的增加,愈伤组织内的木质素含量逐渐降低,而经0.5%浓度硝酸钙处理后的愈伤组织生长慢,生长状态不佳,褐化较严重,处理15d时愈伤组织生长几近停滞。综合比较分析来看,0.1%的硝酸钙浓度对于减少木质素含量有着较为显著的作用。2、基于在10d时愈伤组织在木质素含量上的显著性差异,我们对10d中0.1%硝酸钙处理的愈伤组织,0.5%硝酸钙处理的愈伤组织以及未经过Ca处理的愈伤组织(CK)进行了转录组测序。结果显示,愈伤组织经过不同浓度硝酸钙处理10D后,共确定了2889个差异表达基因,分别在47个GO分组及16个KEGG通路上同时被锚定。此外,在这一过程中我们发现了大量与苯丙氨酸和木质素合成相关的结构基因和转录因子,其中大部分的基因在0.5%硝酸钙处理的情况下发生了显著的上调表达,说明了0.5%的硝酸钙处理活跃了木质素合成的相关转录。在梨愈伤组织施以不同浓度硝酸钙处理后,作为Ca2+受体的钙结合蛋白--钙调蛋白/类钙调蛋白(CAM/CML)、钙依赖性蛋白激酶(CDPK)及钙调磷酸酶B类蛋白(CBL)均有涉及,其中大量的CML基因发生了较大的表达差异。最后,我们选取了苯丙氨酸途径中15个差异倍数较大的差异表达基因及17个梨中CML差异表达基因,并通过实时荧光定量PCR对转录组测序的结果进行验证。结果显示组织上的表达差异和转录组的结果基本一致。3、对梨PbrCML基因进行了全基因组分析,分析结果显示,总计从梨中鉴定出56个PbrCML基因,并根据其结构特征与系统发育将其分为8个亚族。PbrCML基因家族成员分别分布在15条染色体上,共有46个PbrCML基因定位在染色体上,其余10个则基因分布在scaffolds上。共线性分析发现PbrCML基因共有21对共线性基因,且Ka/Ks值表明这21对基因对都经历了纯化选择。通过对PbrCML基因的生物信息学分析,为进一步研究PbrCML基因在梨发育过程中的功能及是否对木质素合成起调控作用提供了资源。4、通过转录组及生物信息学分析推测,PbrCML41可能对木质素合成起着重要作用。荧光定量PCR结果表明,钙调蛋白基因PbrCML41在盛花后45 d的‘砀山酥梨’果实中的相对表达量最高,这与梨果实石细胞与木质素表达规律相符合。PbrCML41亚细胞定位结果表明其是一个膜蛋白。对PbrCML41基因进行功能验证,结果显示,在盛花后35 d的‘砀山酥梨’幼果中瞬时过量表达PbCML41后能引起木质素含量的增加,差异性较显著,果实瞬时注射PbrCML41的区域的表达量相比于未注射区域显著增加。更多还原

【Abstract】 In this experiment,based on the previous studies,the formula of callus medium of’Dangshansu pear’ was further optimized.The pulp of ’Dangshansu pear’ was induced by callus,and the callus of ’Dangshansu pear’ was used as the main material to carry out calcium treatment of different concentrations,combined with the physiological indexes such as lignin content,calcium content,phenotype,etc.,to screen the inhibition wood in order to provide theoretical guidance for the field application of calcium fertilizer to inhibit stone cells.In order to obtain the genes related to calcium binding protein that may be involved in the pathway of calcium regulated lignin synthesis and further explore the regulatory network of calcium affecting lignin formation,we screened out the callus treatment groups with significant physiological differences after calcium treatment and sequenced the transcriptome.Based on the transcriptome data,the candidate gene family PbrCMLs were selected and analyzed,which provided resources for further study on the function of PbrCML gene in Pear development and whether it played a regulatory role in lignin synthesis.Combined with real-time fluorescence q-PCR technology,the key genes obtained from transcriptome data were cloned.Through the technology of instantaneous infection and subcellular localization of pear young fruit,the function of the key genes of pbrcml was verified,providing the molecular basis for revealing the pathway of calcium regulated lignin synthesis.The main results are as follows:1、The medium formulation of Dangshansu pear callus was further optimized,that is,MS 4.74 g/L+1.5 mg/L 2,4-D+0.5 mg/L NAA+0.2 mg/L TDZ+7 g/L agar+30 g/L sucrose+10 g/L sorbitol+0.1 mg/L VC.The callus of ’Dangshansu pear’ was successfully induced and a relatively perfect callus culture technology system was established.Based on the previous field experiments of calcium chloride spraying,the calli of Dangshan pear were treated with different concentrations of calcium nitrate to verify the effect of calcium on the lignin content of calli in vitro,and further explore the appropriate concentration of inhibiting the formation of stone cells.The results showed that calcium nitrate treatment had a certain effect on the calli of pear fruit.The calli treated with 0.1%calcium nitrate had a fast growth rate,a good growth state and a wet surface.With the increase of treatment time,the lignin content in the calli gradually decreased,while the calli treated with 0.5%calcium nitrate had a slow growth,a poor growth state and browning.The growth of callus almost stagnated after 15 days treatment.Comprehensive comparative analysis shows that 0.1%calcium nitrate concentration has a significant effect on reducing lignin content.2、Based on the significant difference in lignin content of callus at 10 days,we sequenced the transcriptome of callus treated with 0.1%calcium nitrate,0.5%calcium nitrate and CK.The results showed that 2889 differentially expressed genes were identified in callus treated with different concentrations of calcium nitrate for 10 days,which were anchored in 47 go groups and 16 KEGG pathways at the same time.In addition,we found a large number of structural genes and transcription factors related to phenylalanine and lignin synthesis in this process,most of which were significantly up-regulated under 0.5%calcium nitrate treatment,indicating that 0.5%calcium nitrate treatment activated the transcription related to lignin synthesis.Calcium binding proteins,CAM/CML,CDPK and CBL,which are Ca2+ receptors,are involved in Pear callus treated with different concentrations of calcium nitrate,among which a large number of CML genes are expressed differently.Finally,we selected 15 differentially expressed genes in phenylalanine pathway and 17 CMLs differentially expressed genes in pears,and verified the results of transcriptome sequencing by real-time fluorescent quantitative PCR.The results showed that the expression difference in tissue was basically the same as that in transcriptome.3、The whole genome of pear PbrCML gene was analyzed.The results showed that 56 PbrCML genes were identified from pear,and they were divided into 8 subfamilies according to their structural characteristics and phylogeny.The members of PbrCML gene family were distributed on 15 chromosomes,46 PbrCML genes were located on chromosomes,and the rest 10 genes were distributed on scaffolds.The results of collinearity analysis showed that there were 21 pairs of collinearity genes in PbrCML gene,and Ka/Ks value indicated that all the 21 pairs had undergone purification selection.Through the bioinformatics analysis of PbrCML gene,it provides resources for further study on the function of PbrCML gene in Pear development and whether it plays a regulatory role in lignin synthesis.4、According to transcriptome and bioinformatics analysis,PbrCML41 may play an important role in lignin synthesis.The results of fluorescence q-PCR showed that the relative expression of calmodulin gene PbrCML41 was the highest in Dangshansu pear fruit 45 days after flowering,which was consistent with the expression of stone cells and lignin in pear fruit.The results of subcellular localization showed that PbrCML41 was a membrane protein.The results showed that the instantaneous overexpression of PbrCML41 in the young fruit of Dangshansu pear 35 days after flowering could increase the content of lignin,and the difference was significant.The expression of PbrCML41 in the area of instantaneous injection of PbrCML41 in the fruit was significantly higher than that in the area without injection.更多还原