摘要
【目的】脂氧合酶（lipoxygenase;LOX;linoleate oxygen oxidoreductase;EC1.13.11.12）作为一种重要的酶,广泛参与植物生长、发育、成熟、衰老及植物抗逆过程,因其重要作用一直是国内外研究的重点和热点。研究利用生物信息学方法对桃LOX基因家族进行发掘和预测,以期为桃LOX家族基因鉴定和功能分析提供基础信息,并在分子水平上为桃品种改良及采后保鲜提供明确的候选基因。【方法】采用生物信息学方法研究了桃LOX基因家族成员数目、系统进化关系、假定蛋白质结构及分类,运用q RT-PCR技术研究LOX基因家族在桃特异组织中的表达模式。【结果】桃LOX基因家族包含13个假定蛋白,9-LOX和13-LOX类型分别有6个成员,另外存在1个特殊类型,与苹果具有相似性;桃LOX蛋白氨基酸序列一致性在33.76%-90.84%;13个LOX家族成员绝大多数是两性氨基酸,9-LOX类型的6个家族成员的理论等电点都小于7,13-LOX类型各等电点没有规律;13个蛋白质的二级结构除ppa017962m外以无规则卷曲为主要构成元件;染色体定位分析表明,LOX家族基因并非平均分布在桃的8条染色体上,其中6号染色体上桃LOX基因分布最多,ppa017962m未定位在任何特异染色体上;q RT-PCR的组织特异性表达结果表明,LOX基因家族主要在果实和茎中的相对表达量较高。【结论】分离并鉴定了13个桃LOX家族基因,其不均匀地分布在桃染色体上;多数桃LOX基因主要在果实和茎部表达;ppa001634m、ppa001082m、ppa001216m、ppa001016m、ppa017962m在果实中的表达量最高,可能参与调控桃果实衰老软化过程。
【Objective】Linoleate oxygen oxidoreductase（LOX; EC1.13.11.12） belongs to a family of non-heme and iron-containing fatty dioxygenases. Lipoxygenase has been reported to play an essential role ina variety of physiological processes, including plant growth and development and plant resistance to abiot-ic stresses, especially in the process of fruit ripening and senescence. In particular, according to priorstudies for LOX, membrane dysfunction associated with LOX through peroxidation of polyunsaturated fat-ty acids could lead to the loss of compartmentalization and cause cells to breakdown, which would directlylead to fruit softening. Moreover, LOX and ethylene together promote the processes of fruit ripening andsenescence, and ethylene treatment enhanced the activity of LOX in fruit. In addition, the products ofLOX have particular importance in tomato fruits, such as participating in the formation of C6 alcohols andaldehydes, which are major volatile flavor components in ripening fruit. LOX are also present in the formof a multi-gene family, in plants. Until now, there are six members of the LOX gene family that were stud-ied in Arabidopsis, 14 members in tomato, and at least 18 LOX-like members in grape. Recently, 23 LOX-like members were identified in either apple or pear. However, studies focusing on the LOX family genesin peaches are relatively rare. It is believed that the multi-member phenomenon of LOX in peaches maybe widespread. However, to our knowledge, mechanisms between the LOX family genes and the ripeningand senescence in perennial woody plants, especially in fruit crops, have not been described in detail. It isa well known fact that the peach fruit is highly perishable in the process of post-harvest storage and itspost-harvest shelf life is very short, which unfavorably affectes the fruit quality and market value. Thisprovides a good reason for research on the relationship between the LOX gene family and the process ofripening and senescence in peach fruit. With the help of a completed peach genome database, it is conve-nient to further identify the molecular mechanism of lipoxygenase, including gene identification and func-tion determination. This study will favorbly provide potential candidate genes for further functional deter-mination, and allow for the comprehension of the expression of the LOX gene family members in differentorgans of the peach, but also provide the theoretical basis for ongoing LOX functional research.【Methods】Hidden Markov Model（HMM） searches were locally performed in the Prunus persica protein databasewhich was obtained from the Genome Database for Rosaceae using the HMM profile of the LOX domain（PF00305） to identify the number of members in the LOX gene family. The amino acid sequences of appleand peach LOX family genes were obtained from the Genome Database for Rosaceae（GDR）. The amino ac-id sequences of the Arabidopsis thaliana LOX family were obtained from the TAIR database. A multiplealignment analysis between the peach and apple and A. thaliana LOX proteins was carried out using theClustal X 2.0.12 program within the MEGA 5 software, and a phylogenetic tree was generated using theneighbor joining method and a bootstrap test was set at 1 000 to test the confidence of the tree. Amino ac-id sequence identity analysis of the LOX family in a peach was performed utilizing the software of DNA-MAN. The online tool, MEME（v4.8.1）, was utilized to search the conversed motifs shared by the LOX pro-teins. The structure of LOX proteins in their physical-chemical properties were analyzed using the onlinetools of Prot Param. The secondary structures of the LOX proteins were predicted by using the HNN Sec-ondary Structure Prediction online tools. The information of the LOX gene family member locations on thechromosomal were searched using the GDR, and the maps were built using the software of Map Inspect.Quantitative real-time PCR（q RT-PCR） were used to analyze the relative expression of the LOX familymembers in different organs or tissues of the‘Xia Hui8＇peach. Specific primers of the peach LOX familygenes for q RT-PCR analysis were designed using a NCBI/Primer-BLAST on-line server.【Results】TheHMM search results showed that the peach genome contained 16 putative LOX gene family proteins whichcontained the LOX conserved domain and that 13 remained after filtering. The Phylogenetic tree of the LOX gene family in peach and apple and A. thaliana showed that the three different species had both simi-larities and differences, all of the three species were primarily classified into 9-LOX and 13-LOX, but itwas also found to be different that the number of amino acid sequences belonged to a specific subgroup.For instance, 6 members belonged to the 9-LOX subgroup and 6 belonged to the 13-LOX subgroup, andthe remaining were classified as special, which was similar to that in the apple but discrepant to A. thaliana. The deduced amino acid sequences of peach LOX members shared an overall identity of 33.76%~90.84%, the identity of ppa001287 m and ppa001311 m were the highest（90.84%）, and the minimum con-sistencies were between ppa001082 m and ppa001016 m. Distribution of the LOX gene family motif inpeach showed that ppa017962 m had one motif missing while the other 12 members had 3 motifs. The pri-mary structure of analysis showed that ppa000968 m had the most number of amino acids andppa017962 m had the least number of amino acids, and most of the peach LOX proteins contained amphi-philic amino acids. Six members of the 9-LOX subgroup had a theoretical isoelectric point of less than 7,and there was no regularity among members of the 13-LOX subgroup. The second structure for all the putative peach LOX proteins was mainly a random coil, with the exception of ppa017962 m. The LOX gene family member locations on the peach chromosome were asymmetric, and the maximum LOX gene family members were located on the sixth chromosome, and four paralog genes were particularly located on it.Notably, the ppa017962 m gene was not located on any chromosomes, which may indicate that it floats in the peach genome. Tissue specific expression determination via q RT-PCR analysis showed that the peach LOX family genes were primarily expressed in fruits and stems. Particularly, genes of the ppa001634 m were expressed higher than other LOX gene family members in stems, leaves and flowers respectively. In addition, the genes of ppa001082 m, ppa001216 m, ppa001016 m, and ppa017962 m were primarily expressed in peach fruits.【Conclusion】In this study, thirteen LOX family genes were isolated and characterized in peaches, which were unevenly distributed in the peach genome. The LOX family genes were primarily expressed in fruits and stems. Importantly, ppa001634 m, ppa001082 m, ppa001216 m, ppa001016 m,and ppa017962 m had the highest expression level in fruit among the different organs of the peach, implying that these genes may play a large role in the peach fruit softening process.
出处
《果树学报》
CAS
CSCD
北大核心
2016年第3期257-267,共11页
Journal of Fruit Science
基金
江苏省农业科技自主创新基金项目[CX(15)1020]
国家现代农业产业技术体系建设专项资金(CARS-31)
