一个棉花GDSL脂肪酶基因的克隆与功能分析

摘要

GDSL and GXSXG lipases are two important sub-families of lipases.Unlike the GXSXG motif-containing lipases,GDSL lipases have a GDSL motif and are active in hydrolysis and synthesis of abundant ester compounds.In this experiment,a EST sequence (GenBank accession number:DR458916) was screened based on extremely significant expression differences at fiber initiation stages between lintless-fuzzless XinWX and linted-fuzzless XinFLM isogenic lines by 29K cotton genome array hybridization.Using the EST sequence as queries,the Gossypiurn EST database (http://www.ncbi.nlm.nih.gov/) was screened and the corresponding cDNA sequences containing a complete ORF were assembled.Further,the ORF information was reconfirmed in transcriptional and genomic level.As a result,a novel gene encoding GDSL lipase was cloned,and the gene was designated as GhGDSL (Gossypium hirsutum GDSL; GenBank accession number:KC186125).GhGDSL included an open reading frame of 1065 bp that encoded a polypeptide of 354 amino acids.The genome sequence indicated that GhGDSL had four introns and five exons.The homolog of GhGDSL had one copy in diploid cotton species G.herbaceum and G.raimondii and two copies in tetraploid cotton species G.hirsutum acc.TM-1 and G.barbadense cv.Hai 7124,respectively.Sequence alignment from different cotton species showed GhGDSL homoelogs in tetraploid were evolved independently,with more variations in D-subgenome than in A-subgenome.GhGDSL homoelogs in tetraploid were located on chromosome 4 (Chr.A4) and chromosome 22 (Chr.D4) by developing subgenome-specific SNP marker,respectively.Q-PCR analysis showed that GhGDSL was accumulated highly in ovule and fiber tissue at 3-10 days post anthesis (DPA),with expression peak at 8DPA in Hai 7124 and high expression from 5DPA to 10 DPA in TM-1.The association analysis between GhGDSL and the traits related to fiber and seed qualities in corresponding [(TM-1 ×Hai 7124)×TM-1] BC1S1 individuals showed that GhGDSL homolog in A-subgenome was significantly correlated with the seeds' fat content (P=0.048),and GhGDSL homolog in D-subgenome was very significantly correlated with the seeds' protein content (P=-0.008).These results suggested that GhGDSL might be functionally important in the metabolic process of lipid and protein in seeds and in the process of fiber development.%GDSL脂肪酶与GXSXG脂肪酶是2个重要的脂肪酶亚家族.其中,GDSL家族脂肪酶具有水解酶活性,能水解多种酯类物质.本试验根据新乡小吉无绒无絮(XinWX)和无绒有絮(XinFLM)近等基因系纤维起始期29K芯片竞争杂交结果,选择了一个在纤维起始期具有极显著表达差异的EST序列(GenBank登录号为DR458916),以该序列信息为探针,利用电子克隆方法并进行cDNA及基因组全长基因PCR扩增、测序验证,克隆获得一个陆地棉GDSL脂肪酶基因(GhGDSL; GenBank登录号为KC186125).该基因ORF长1065 bp,编码354个氨基酸,含有5个外显子和4个内含子.该基因在二倍体棉种基因组中含一个拷贝,在四倍体棉种基因组中含2个拷贝.序列比对显示该基因在四倍体棉种的2个亚组中独立进化,且D亚组比A亚组变异大.染色体定位显示该基因2个拷贝分别位于A4 (Chr.4)和D4 (Chr.22)染色体上.定量RT-PCR结果表明,GhGDSL在开花后3～10 d的纤维组织中表达量高,其中在海7124中表达高峰在8DPA,在TM-1中表达高峰从5DPA持续到10DPA.利用[(TM-1×Hai7124)×TM-1]的BC1S1群体开展Gh GDSL功能与纤维、种子品质性状关联分析,发现该基因A亚组的拷贝与种子脂肪含量存在显著相关(P=0.048);D亚组的拷贝与种子蛋白含量存在极显著相关(P=0.008).推测Gh GDSL基因功能与种子中脂肪、蛋白代谢相关,同时也参与纤维伸长过程.