沙枣叶绿体全基因组序列及其使用密码子偏性分析

该研究以2株野生沙枣(Elaeagnus angustifolia Linn.)嫩枝经温室水培后的嫩叶为材料,采用CTAB法分别提取总DNA,并利用第二代测序技术进行总DNA从头测序,组装后得到2株沙枣叶绿体基因组全序列,并详细分析了其蛋白质编码基因密码子使用的偏好性及其原因,为沙枣叶绿体基因工程和分子系统进化等研究奠定基础。结果显示:(1)组装得到沙枣叶绿体基因组序列全长150 546 bp,由长度为81 113 bp的长单拷贝(LSC)区域和25 494 bp的短单拷贝(SSC)区域,以及1对分隔开它们的长18 445 bp的反向重复序列(IRS)组成;注释共得到132个基因,包括86个蛋白编码基因、38个tRNA基因和8个rRNA基因。(2)沙枣叶绿体基因组蛋白编码基因密码子的第三位碱基GC含量(GC_3)为28.47%,明显低于整个叶绿体基因组GC含量(37%),也低于第一位(GC_1)和第二位(GC_2)碱基的GC含量,说明密码子对AT碱基结尾有偏好性;其中, UCU、CCU、UGU、GCU、CUU、GAU、UCA和UAA为最优密码子。(3)同义密码子相对使用频率(RSCU)分析发现,影响密码子使用模式的因素并不单一,密码子的偏好性受到突变、选择及其他因素的共同影响,并且自然选择表达引起的序列差异比突变对密码子偏好性的影响要显著;中性绘图分析、有效密码子数(ENC-plot)分析和奇偶偏好性(PR2-plot)分析表明,沙枣叶绿体基因组使用密码子的偏性受选择的影响更大。(4)通过最大似然法、最大简约法和贝叶斯方法对胡颓子科6个物种和1个枣的叶绿体基因序列构建系统发育树,与它们使用密码子偏性聚类的结果一致,表明叶绿体基因组使用密码子偏性与物种的亲缘关系相关。

Assembling and Analysis of the Whole Chloroplast Genome Sequence of Elaeagnus angustifolia and Its Codon Usage Bias

In this study, the whole chloroplast genome sequence of Elaeagnus angustifolia Linn was assembled by sequencing total genome DNA with the second generation sequencing. Young leaves of E. angustifolia were collected from shoots of two wild individuals under hydroponic growth in greenhouse , and total DNA of the two individuals were extracted separately by CTAB method for two repeats of the chloroplast genome. The codon usage bias of protein coding genes and its causes were analyzed in detail. The results of this study are of great significance for future chloroplast genetic engineering and molecular phylogenetic researches in E.angustifolia. The results show: (1) the full chloroplast genome sequence of E. angustifolia was a circular molecule of 150 546 bp in length with a typical quadripartite structure, including a pair of inverted repeats (IRA and IRB) of 25 494 bp that were separated by large and small single copy regions (LSC and SSC) of 81 113 bp and 18 445 bp, respectively. A total of 132 predicted genes, including 86 protein coding genes, 39 tRNA genes and 8 rRNA genes, were identified. (2) The GC content of the third nucleotide in all protein coding genes was 28.47%, which was significantly lower than that GC₂ and GC₁, indicating that the E. angustifolia chloroplast gene codon has a preference for the end of the A or T base, and eight optimal codons, UCU, CCU, UGU, GCU, CUU, GAU, UCA and UAA were determined. (3) We analyzed the codon bias of the chloroplast genomic protein coding sequence of E. angustifolia. Corresponding analysis of RSCU indicated that mutations, selection and other factors may all affect the codon usage pattern, and the sequence difference caused by natural selection is more significant than the mutation to codon usage bias. Effective Number of Codon plot (ENC plot) analysis and Parity rule 2 plot (PR2 plot) analysis indicated that the bias of codons in the chloroplast genome of E. angustifolia was mainly influenced by selection, although mutation may also have played a role. (4) Cluster tree based on the Relative Synonymous Codon Usage (RSCU) values of chloroplast genes from seven species in Elaeagnaceae is basically consistent with phylogenetic tree based on chloroplast whole genome sequences, which illustrated that codon usage bias pattern in the chloroplast genomes of species in Elaeagnaceae was related to the phylogenetic relationship of these species.