【作者】 陈杰；

【导师】 彭定祥；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2017， 博士

【摘要】 苎麻（Boehmeria nivea L.Gaud）是荨麻科（Urticaceae）苎麻属（Boehmeria）的多年生宿根性草本开花植物。苎麻主要采用传统育种方法进行育种,由于种植面积大幅萎缩、科研人员不足以及研究基础薄弱等原因,其新品种的育种进程一直比较缓慢,需要尽快引入分子育种手段加快其育种进程。本研究对苎麻纤维发育进行了转录组测序和对expansin家族进行了基因克隆和功能分析,主要实验结果如下:1.进一步优化了苎麻不同样品总RNA提取技术,并使用该基于异硫氰酸胍作为主要裂解成分的提取技术,成功从苎麻不同部位茎皮材料分别提取得到高质量总RNA,以用于转录组测序。转录组测序分别使用黑杆前期苎麻不同部位茎皮材料代表韧皮纤维不同发育时期,连接不同接头之后使用罗氏454 FLX+平台进行混样转录组测序,共得到1030057个reads,平均读长457 bp。对测序结果进行拼接后,共得到58369条unigene,序列长度范围为90 bp到7641 bp,其中51025条（占87.42%）序列长度大于200 bp,8025条（占13.75%）序列长度大于1000 bp。最后,58369条unigene（13386条contig和44983条isotig）中有25071条unigene（7065条contig和18006条isotig）得到了有效的注释,共对应于6283条GO条目和3076条KO条目。通过对差异性表达基因、GO条目富集情况等分析表明茎皮上部样品在所有的样品中更为特殊,其所对应的苎麻纤维发育早期可能更容易受分子调控影响,从而影响苎麻纤维发育状况。在这之中,属于纤维素合酶基因家族的isotig01514₇7、isotig06919₇8、isotig06943₂5和isotig07154₁9,属于expansin基因家族的isotig10345₂3、isotig01251₆和isotig02054₁8,以及属于XTH基因家族的HRX1MBH01BLWDW₉、isotig00610₂5、isotig01663₂2、isotig04660₈、isotig09773₁0和contig04902₁9在该样品相对于其余三个样品均为上调表达,表明这些基因可能参与苎麻茎皮上部样品中针对纤维发育的相关调控路径。2.使用同源序列克隆的方法,获得了五条苎麻expansin基因全长编码序列,并对表达量进行了初步研究。基于本实验室的三个转录组测序信息,克隆得到的expansin基因扩充至16个（12个属于α-亚族,分别命名为BnEXPA1-BnEXPA12;4个属于β-亚族,命名为BnEXPB1-BnEXPB4）,共有22条（其中6个expansin基因在基因组中均有两个序列相异的克隆,分别命名为-1和-2）全长DNA序列（GenBank登录号:KY748166-KY748187）。序列分析表明,其外显子长度跨度较小（107 bp-485 bp）而内含子长度跨度较大（86 bp-1540 bp）,cDNA全长分布为732 bp-831 bp,而DNA全长跨度为1040 bp-4259 bp。在随后对所克隆得到的expansin基因进行表达分析中,BnEXPA2、BnEXPA3、BnEXPA5和BnEXPA6在六个样品（茎尖、幼叶、成熟叶、茎皮上中下部）中的整体表达量和其它基因相比较高,BnEXPA12在这六个样品中表达量差异性最大,BnEXPA3、BnEXPA5、BnEXPA10和BnEXPB2在苎麻三季麻（头麻、二麻和三麻）不同时期（苗期、快速生长期、纤维成熟前期和后期）茎皮不同部位（苗期下部茎皮、快速生长期上部和下部茎皮、纤维成熟期上部、中部和下部茎皮）样品中的表达量和其它基因相比较高,BnEXPA11在上述所有样品以及三种胁迫处理（氯化钠、高温和低温胁迫）条件下表达量均极低。最后,BnEXPA1在不同胁迫条件下其表达量均显著上升,也是所有苎麻expansin基因中唯一一个在三种胁迫条件下相对表达量均显著上升的基因。与之相反,BnEXPA4、BnEXPA5、BnEXPA12、BnEXPB1和BnEXPB3在三种胁迫条件下其表达量均显著下降。这些在表达量研究过程中呈现一定表达规律的基因都可作为候选基因在后续实验中优先得到进一步的详细研究。综上,本项研究主要亮点如下:1.优化了苎麻RNA提取技术,并用于转录组测序中;2.转录组测序取样方法为苎麻中首创,与纤维发育状态对应;3.首次对苎麻茎皮样品进行转录组测序,研究纤维发育分子机理;4.使用同源序列克隆方法得到五条苎麻expansin基因序列;5.整合多个转录组测序结果进行苎麻expansin家族基因克隆;6.得到一批可能参与苎麻抗逆以及纤维发育相关通路的expansin基因。以上结果既对苎麻纤维发育分子机理进行了初步研究,也为后续苎麻基因工程以及相关领域研究提供了范式和平台数据。更多还原

【Abstract】 Ramie（Boehmeria nivea L.Gaud）is a perennial herbaceous flowering plant.Due to the significant shrink of ramie planting area,insufficient manpower in scientific research,and less research accumulation,the new cultivar breeding progress of ramie,which was mostly conducted through traditional breeding methods,went slowly.It is urgent to introduce current molecular breeding tools into ramie breeding processes to accelerate the progress.In this study,researches of ramie transcriptome sequencing regarding different fiber developmental status,cloning and expression profiling analysis of expansin gene family in ramie were conducted.The main results were as following:1.The total RNA extraction methods were further optimized,the high quality total RNA,which was utilized in the transcriptome sequencing,was isolated using the improved guanidinium-based protocol.Different parts of ramie bark samples were generated to represent different ramie fiber developing stages.After ligating to distinguishable adaptors,RNAs isolated from these samples were equally mixed and performed a full run sequencing on the Roche 454 FLX+ titanium platform.A total of 10,300,057 reads with average length of 457 bp were obtained.After assembling,the sequence lengths of 58,369 unigenes ranged from 90 bp-7641 bp,in which 51,025 sequences（87.42% of total unigenes）with lengths over 200 bp,and 8,052（13.75%）with lengths over 1000 bp.Finally,25,071 unigenes（7065 contigs and 10,886 isotigs）out of 58,396 unigenes（13,386 contigs and 44,983 isotigs）were efficiently annotated,corresponding to 6283 GO entries and 3076 KO entries.After analyzing multiple items,like the differentially expressed genes and GO entry enrichment,we assumed that the top part of ramie bark sample is special among all the four samples,which means the early developmental stage is more sensitive to molecular regulation and thus affecting ramie fiber development.Among the differentially expressed genes in the particular sample,the unigenes isotig01514₇7,isotig06919₇8,isotig06943₂5 and isotig07154₁9 from the cellulose synthase gene family,the isotig10345₂3,isotig01251₆ and isotig02054₁8 from the expansin gene family,and the HRX1MBH01BLWDW₉,isotig00610₂5,isotig01663₂2,isotig04660₈,isotig09773₁0 and contig04902₁9 from the XTH gene family,were up-regulated when comparing to the rest three samples,indicating these unigenes were probably involved in the pathways regulating ramie fiber development in the upper part of ramie stem bark sample.2.Using the homologous sequence cloning strategy,we cloned five expansin genes with full coding sequences from ramie,expression analyses were also conducted.After searching the three transcriptome libraries with keywords ‘expansin’ in annotation information,the number of cloned expansin genes was expanded to 16（12 of them belong to α-subfamily,the rest 4 belong to β-subfamily）,with 22（Gen Bank accession: KY748166-KY748187）full-length DNA sequences（each of 6 expansin genes has two clones with different sequences in ramie genome,which was respectively named as-1 and-2）.For sequence length variation of the 22 cloned sequences,exons varied from 107 bp to 485 bp,while the introns varied from 86 bp to 1540 bp;the lengths variation for c DNA was 732 bp to 831 bp,while for DNA was 1040 bp to 4259 bp.Afterwards,the expression analysis for the cloned expansin genes were conducted.Bn EXPA2,Bn EXPA3,Bn EXPA5 and Bn EXPA6 expressed generally higher than other expansin genes in the six samples（shoot apex,young leaves,mature leaves,the top,middle and bottom part of stem bark）of ramie,while Bn EXPA12 expressed most differently among these six samples.Bn EXPA3,Bn EXPA5,Bn EXPA10 and Bn EXPB2 expressed generally higher among the samples obtained from different ramie stem bark samples（bottom part at seedling stage,top and bottom parts at rapid growing stage,top,middle,and bottom parts at fiber mature stages）in the three growing seasons of ramie,Bn EXPA11 expressed extremely low in all samples and under advert conditions（Na Cl,high temperature and low temperature stresses）.At last,Bn EXPA1 was up-regulated under any of the three abiotic conditions,which was the only one like this in the expansin family of ramie.On the contrary,Bn EXPA4,Bn EXPA5,Bn EXPA12,Bn EXPB1 and Bn EXPB3 were down-regulated under the three advert conditions.These genes showing special expression patterns can be regarded as candidate genes in downstream experiments for further detailed analysis.All in all,there have the following highlights in this study:1.RNA isolation methods were further optimized and applied in the following transcriptome sequencing of ramie;2.It was the first time to use the sampling method stated here,which represented different fiber developmetal stages in ramie,for transcriptome sequencing;3.It was the first time to use the high-throughput sequencing platform to illuminate the molecular mechanisms of ramie fiber development;4.Five expansin genes were cloned utilizing the homologous sequence cloning strategy;5.Expansin gene cloning was expanded by combining multiple transcriptome sequencing data;6.A bunch of expansin genes were presumably included in the pathways corresponding to advert conditions or fiber developmental processes of ramie.In a word,the molecular mechanisms of ramie fiber developmemt were preliminarily studied,which provided experimental data and operational mimic for downstream studies.更多还原