【作者】 张波；

【导师】 陈昆松；

【作者基本信息】 浙江大学 ， 果树学， 2007， 博士

【摘要】 以美味猕猴桃(Actinidia deliciosa)果实为试材，开展了LOX基因家族成员克隆，并对其进行生物信息学分析，建立了基于LOX基因家族成员表达的QPCR技术体系，研究各成员在不同器官、果实生长发育和成熟衰老进程中的表达模式，以及不同处理(温度和乙烯)对LOX基因的表达调控，鉴别了猕猴桃果实后熟软化和香气形成相关家族成员。主要结果如下：1、利用猕猴桃EST库及相关生物信息学手段，克隆了LOX基因家族成员6个，命名为AdLox1、AdLox2、AdLox3、AdLox4、AdLox5和AtLox6，编码区长度分别为2739、2595、2739、2709、1359和1557bp，其中，AdLox1、AdLox2、AdLox3和AdLox4为全长cDNA序列。猕猴桃LOX基因家族成员的序列同源性存在差异，AdLox2和AdLox5氨基酸序列同源性最高，约74％，而AdLox3和AdLox5仅为49％。生物信息学分析表明，AdLox1、AdLox3、AdLox4和AdLox6可能具有13-LOX活性，而AdLox2和AdLox5则属于9-LOX类型，它们均具有保守的38个氨基酸残基，但13-LOX在N端含有额外约60个氨基酸残基。猕猴桃AdLox1与番茄TomLoxD具有最高氨基酸序列同源性(80％)，AdLox2和AdLox5与TomLoxA高度聚类，序列同源性分别为68和75％，AdLox4和AdLox6与TomLoxC的氨基酸序列同源性分别为62和63％，AdLox3与其它植物LOX序列同源性低于55％。2、完善并建立了基于LOX基因家族成员功能研究的QPCR技术体系，该体系具有良好稳定性和重复性，针对基因家族成员序列差异设计的特异性引物，有效地避免了交叉扩增污染，提高了基因表达检测的准确性。3、猕猴桃AdLox1在根、茎和花中表达水平接近；AdLox2转录本主要在根、茎和叶中积累，且水平相近；AdLox3、AdLox4和AdLox6表达分布类似，主要存在于茎和叶；AdLox5在根中具有较高表达水平，但绝对转录本很低。果实生长发育进程中，AdLox1、AdLox2、AdLox3和AdLox6具有相似的表达模式，其转录本水平从20至80daa呈下降趋势，在100daa则略微增强，然后再次下降直至果实采收；AdLox4的表达水平在果实发育进程中则维持基本稳定，但转录本水平较高；AdLox5表达水平很低。4、20℃下猕猴桃果实成熟衰老进程中，AdLox1和AdLox5表达随乙烯积累呈增加趋势，而AdLox2、AdLox3、AdLox4和AdLox6转录本在果实后熟软化过程中趋于下降。外源乙烯处理(100μl／L，20℃，24h)显著加速果实后熟软化进程，诱导LOX活性上升和MDA含量增加，促进了AdLox1和AdLox5表达水平增强，而AdLox2，AdLox3，AdLox4和AdLox6的转录本则被明显抑制，在处理24h内分别下降了5、20、10和2倍。烟草叶片的瞬时表达结果表明，AdLox1显著加快叶绿素降解(P＜0.05)，促进叶绿素荧光下降(P＜0.05)，诱导组织衰老，而AdLox2则与野生型对照植株无显著差异。5、0℃低温处理168h，猕猴桃果实硬度和TSS维持稳定水平，转入20℃货架期贮藏后，成熟进程显著加快。0℃贮藏过程AdLox1、AdLox5和AdLox6表达水平逐渐增强并72h左右达到高峰，随后下降，这一趋势与LOX活性变化相吻合。AdLox2、AdLox3和AdLox4转录本水平在低温贮藏期间维持稳定。6、LOX活性在猕猴桃果实成熟进程中呈峰型变化(约180h出现高峰)，伴随有己醛和(E)-2-己烯醛含量减少。至20℃贮藏276h，果实(E)-2-己烯醛水平比180h时下降了约7倍。LOX基因家族AdLox2、AdLox3、AdLox4和AdLox6的表达趋势与猕猴桃果实香气物质变化相一致。果实圆片组织的离体试验表明，1.0mmol／L LA和1.0mmol／L LeA处理显著促进LOX活性(P＜0.05)，诱导AdLox4和AdLox6表达增强，其中AdLox6的转录本分别增加了12和6倍，而AdLox2和AdLox3表达水平基本不变。上述研究结果表明，LOX基因家族在猕猴桃果实生长发育和成熟衰老进程中具有表达差异，对温度和乙烯处理表现出不同的应答模式。更多还原

【Abstract】 Studies of LOX gene family members function and regulation were carried out by using postharvest kiwifruit (Actinidia deliciosa) as plant materials. The main results are as followings.From the HortResearch Actinidia EST database, we identified and cloned six LOX genes, i.e. AdLox1-6. AdLox1, AdLox2, AdLox3 and AdLox4 contained full-length cDNAs with 2739, 2595, 2739 and 2709 bp respectively, AdLox5 and AdLox6 were cDNA fragments of 1359 and 1557 bp. Among the six kiwifruit LOX genes, AdLox5 and AdLox6 had the highest amino acid dequence identity, which was 74%, while AdLox3 and AdLox5 produced the lowest amino acid sequence identity, which was 49%. Phylogenetic analysis showed that AdLox2, AdLox5 clustered as 9-LOX, while AdLox1, AdLox3, AdLox4 and AdLox6 clustered as 13-LOX. There is a signal peptide sequence of 60 amino acids in all members of the 13-LOX group, but no similar sequence is found in the 9-LOX genes. AdLox1 was most similar to TomLoxD at 80% identity at the amino acid level, AdLox2 and AdLox5 matched to TomLoxA with 68 and 75% identity respectively, and AdLox4 and AdLox6 showed 62 and 63% sequence identity to TomLoxC, however, AdLox3 had less than 55% identity to LOX genes from tomato.Closely related genes that were very similar at the sequence level, such as AdLox2 and AdLox5 had 74% identity at amino acid level, and therefore, it may be difficult to determine the RNA level of a specific LOX gene member. This problem is resolved by the high specificity of real-time quantitative PCR (QPCR) using of specific oligonucleotide primers. The primers were designed at the 3’UTR, which was divergent among the LOX gene family members. The primers showed no cross-amplification to other members of the family. End-point semi-quantitative PCR (SQPCR) was used to examine the expression pattern of the six LOX genes in root, stem, leaf and petal tissues, and during fruit development. AdLox1 had similar transcript abundances in root, stem and petal; AdLox2 expressed mainly in kiwifruit root, stem and leaf; AdLox3, AdLox4 and AdLox5 had similar distributions, and expressed mainly in stem and leaf; transcripts of AdLox5 was barely detectable in the various tissues. During fruit development, young fruit at 20 days after anthesis (daa) had relatively high expression levels of LOX genes, similar to those observed in stem tissues. AdLox1, AdLox2, AdLox3 and AdLox6 exhibited similar expression patterns throughout fruit development, with transcript abundance decreasing from 20 to 80 daa and increasing again at about 100 daa. AdLox4 showed a relatively constant expression level during fruit development, and produced the strongest bands among the LOX gene family. AdLox5 transcript was almost undetectable during fruit development.For fruit stored at 20℃without ethylene treatment, transcripts of AdLox1 and AdLox5 increased with kiwifruit ripening. In contrast, expression of AdLox2, AdLox3 AdLox4 and AdLox6 decreased when kiwifruit ripening progressed to the climacteric stage. During ethylene treatment (100μl/l, 24 h, 20℃), there was an increase in total LOX enzyme activity. After the ethylene treatment, LOX activity returned to harvest levels within 24 h and subsequently increased again as fruit ripened further. MDA accumulated gradually with fruit ripening. The relative QPCR results showed that expression of AdLox1 and AdLox5 was stimulated by ethylene treatment within 24 h, however, transcript abundance of AdLox2, AdLox3, AdLox4 and AdLox6 decreased about 5-, 20-, 10- and 2-fold, respectively, within 24 h of exposure to ethylene. Over-expression of AdLox1 in tobacco leaves significantly accelerated tissue chlorophyll degradation (P<0.05) and decreased chlorophyll fluorescence (P<0.05). However, over-expression of AdLox2 had no effect on senescence as measured by these two properties.Kiwifruit held at 20℃, there was an increase in LOX enzyme activity and peaked at about 180 h after harvest, following a significant decrease (P<0.05) during fruit ripening and senescence. Fatty acid-derived C6 aldehydes, such as hexanal and (2s)-2-hexenal, which are derived from a LOX-mediated pathway, give the kiwifruit a green aroma character. Amount of the two compounds trended to decrease during kiwifruit ripening, especially for (E)-2-hexenal, which showed 7 fold declines from 180 to 276 h after harvest. Along with the time when there was a decrease in C6 aldehydes, expression levels of AdLox2, AdLox3, AdLox4 and AdLox6 showed a significant reduction. Treating kiwifruit flesh discs with LA and LeA resulted in a significant increase in LOX activity (P<0.05), and marked transcripts accumulation in AdLox4 and AdLox6. Transcripts of AdLox6 were induced by 12- and 6-fold in response to LA and LeA, respectively. In contrast, AdLox2 and AdLox3 maintained constant expression level in treated tissues.Fruit held at low temperature (0℃, 168 h) maintained firmness, and did not significantly soften after transfer to 20℃for 72 h. Transcripts of AdLox1 were strongly induced by the low temperature treatment, peaking at about 72 h after harvest, and this expression declined during shelf life. AdLox5 and AdLox6 had similar expression patterns to those of AdLox1 during the low temperature treatment. The expression profiles of the above three LOX genes matched changes in LOX enzyme activity in response to low temperature. However, AdLox2, AdLox3 and AdLox4 showed no significant changes in transcript levels during low temperature treatment.These results showed that the LOX gene family members were differentially regulated during kiwifruit ripening and senescence, and they showed different response to temperature and ethylene treatment. The possible roles of individual LOX isoforms in kiwifruit were discussed.更多还原