Abstract: This study investigates the codon usage bias in of Trifolium pratense L.,laying the foundation for optimizing codon composition. The codon usage patterns and bias of 5 513 coding sequences of Trifolium pratense leaf transcriptome were analyzed using software such as CodonW,R,and CUSP. The study indicated that there was a weak correlation between GC3 and GC12,indicating that selection pressure plays a certain role in codon usage bias. In the ENC-plot analysis,most codons showed relatively weak usage bias,suggesting that selection pressure was the main driver of bias,while a few genes were evenly distributed on both sides of the expected curve,indicating the influence of mutational pressure on some genes. CAI analysis revealed that gene expression levels had a certain impact on the process of codon usage bias,with a CAI of 0.201 indicating relatively weak bias. Biased analysis further confirmed that codon usage bias in Trifolium pratense was mainly influenced by mutational pressure. Analysis of optimal codons showed that the optimal codons in the transcriptome of Trifolium pratense preferentially end with G or C. These results are beneficial for optimizing codon usage in genetic engineering studies of Trifolium pratense and are of significant importance for the systematic evolution research of Trifolium pratense.

Key words: Trifolium pratense leaves, Codon preference, Transcriptome, Encoding sequence

摘要： 研究红三叶(Trifolium pratense L.)叶片的密码子使用偏好性，可为优化密码子组成奠定基础。研究通过CodonW，R，CUSP等软件对红三叶叶片转录组中5 513条编码序列进行密码子使用模式和偏好性分析。研究发现，GC3与GC12相关性较弱，表明选择压力对其密码子偏好性的选择有一定的作用；ENC-plot分析中，大多数密码子偏好性相对较弱，表明选择压力是偏好性的主要来源，少数的基因在期望曲线两侧均衡散布，表明部分基因受突变压力的作用；密码子适应度值(Codon adaptation index，CAI)分析可知，基因表达水平对密码子偏好性产生过程具有一定影响作用，CAI为0.201，表明密码子偏好性比较弱。偏倚性分析进一步证实红三叶叶片密码子偏好性重点受突变压力的影响。最优密码子分析表明，红三叶叶片转录组序列的最优密码子更偏好以G或C结尾。结果有利于红三叶基因工程研究中密码子优化，对红三叶的系统进化研究具有重要意义。

关键词: 红三叶叶片, 密码子偏好性, 转录组, 编码序列