基于马尾松干旱转录组的抗旱功能SSR位点分析

Analysis of SSR Loci of Functional Gene Linked to Drought Resistance Based on Transcriptome Sequences in Pinus massoniana under Drought Stress

[目的]了解马尾松干旱胁迫转录组序列的功能分布和SSR位点分布特征,并探索与干旱关联的SSR位点。[方法]对马尾松幼苗进行持续干旱胁迫,选取干旱10、15、25 d及正常供水对照的马尾松针叶样品,通过总RNA提取、Illumina测序、raw reads去冗、Trinity拼接获得unigenes。利用Blast比对,对Unigene序列进行GO、KOG、KEGG功能注释及分类;利用Misa软件进行SSR位点批量搜寻,primer 3. 0软件进行规模化SSR引物设计;利用GOSeq(1. 10. 0)、KOBAS(v2. 0. 12)软件对差异表达的含SSR位点Unigene进行GO、KEGG显著性富集分析。[结果]马尾松转录组194 821个Unigene中,101 806个Unigene获得注释,包含64 943个GO功能注释、35 880个KOG功能注释及30 882个KEGG注释。搜寻到6 728个SSR位点,分布于6 367个Unigene中,SSR出现频率为3. 45%;重复类型以单、三、二核苷酸为主,分别占总SSR的35. 82%、33. 03%和25. 22%;重复基序以A/T、AT/AT、AG/CT、AGC/CTG、AAG/CTT为主;基序长度以10 20 bp的短序列SSR为主;基序重复次数以5～10次重复占优势;批量设计13 338对SSR引物。422个含SSR位点Unigene具有差异表达; KEGG富集分析发现,有11个含SSR位点的差异Unigene参与了光合作用、植物激素信号传导及类胡萝卜素合成等3个与干旱响应相关的代谢途径。[结论]从较高质量的马尾松转录组中获得101 806个具有注释的Unigene;从6 367个Unigene中挖掘出6 728个SSR位点; 422个含SSR位点的差异表达Unigene中筛选出11个与干旱关联的SSR功能位点,为抗旱功能基因定位及马尾松抗旱分子机制研究奠定基础。

[Objective]The transcriptome data of Pinus massoniana under drought stress were used to clarify the function distribution of sequences,as well as the characteristics and distribution patterns of SSR loci,and to explore the key SSR loci linked to drought-resistant gene. [Method] The P. massoniana needle samples under lingering drought stress for 10,15,and 25 days and the corresponding samples with sufficient water as the control（ CK） were selected to extract the total RNA. Illumina sequencing were performed to generate raw reads. After removal of lowquality data,the transcriptome assembly was conducted using Trinity software. The unigenes of transcriptome were annotated by aligning with several public databases via BLAST program,including GO（ Gene Ontology）,KOG（ eukaryotic orthologous groups）,and KEGG（ Kyoto Encyclopedia of Genes and Genomes）. The SSRs loci were ex-amined using Misa software,and the PCR amplification SSR primers were designed using Primer 3. 0 software. GO and KEGG enrichment analysis were implemented using GOSeq（ 1. 10. 0） and KOBAS software,respectively,to determine the major process of biological process and metabolic pathways of differentially expressed unigenes contained SSR loci. [Result]A total of 101 806 unigenes were annotated from 194 821 unigenes of transcriptome. Among them,64 973 functional annotations were from GO database,35 880 from KOG database and 30 882 from KEGG database. Moreover,6 728 SSR loci were identified and distributed in 6 367 unigenes,and their average frequency of SSRs was 3. 45%. Among all the SSR motifs,mononucleotide,trinucleotide and dinucleotide were the major repeated types,with occurrence frequency of 35. 82%,33. 03% and 25. 22%,respectively; the form of A/T,AT/AT,AG/CT,AGC/CTG,and AAG/CTT were the most frequent motifs,the length from10 to 20 bp were the most repeat motifs,and the SSR repeat numbers from 5 to 10 were the most repeat numbers of motifs. A total of13 338 pairs of SSR primers were designed for marker development of P. massoniana. Furthermore,among the6 367 unigenes containing SSR loci,422 unigenes were differentially expressed on drought stress versus the control.Enriched analysis of KEGG pathway showed that 11 unigenes containing SSR loci were significantly enriched into three KEGG pathways,including photosynthesis,plant hormone signal transduction and carotenoid biosynthesis,which were linked to the plant response to drought stress. [Conclusion]A total of 101 806 unigenes were annotated from a higher quality of transcriptome database in P. massoniana,6 728 SSR loci were identified and distributed from 6 367 unigenes,11 SSR loci from 422 differentially expressed genes containing SSR loci were identified linking to the plant response to drought stress. These results can be used for the subsequent study on molecular mechanism for drought resistance and functional gene localization in P. massoniana.