基于RNA-seq的套袋对‘天源红’猕猴桃果实花色苷合成关键结构基因表达的影响

Effect of bagging on expression of crucial structural genes of anthocyanin biosynthesis in 'Tianyuanhong' kiwifruit based on RNA-seq

【目的】探讨套袋对红肉猕猴桃果实生长发育过程中不同部位花色苷合成关键结构基因表达的影响,为解析光照对花色苷合成分子机制的影响提供依据。【方法】以全红型软枣猕猴桃‘天源红’为试材并对其进行套袋处理,在RNA-seq的基础上,筛选候选基因,利用实时荧光定量PCR对这些候选基因在‘天源红’盛花后7个时期的果肉样品中的表达量进行相对定量。根据荧光定量结果和聚类分析,最终确定关键候选基因,利用实时荧光定量PCR对关键候选基因在‘天源红’不同处理、不同时期、不同果实部位中的表达量进行相对定量分析,并对关键候选基因的表达量和花色苷含量进行相关性分析。【结果】12个候选基因在盛花后7个时期中的表达具有一定的规律,且各自的表达规律不尽相同。在果肉颜色明显变为红色的盛花后110 d,F3H、LDOX和F3GT的表达量较其他时期高。聚类分析结果显示,LDOX和F3GT分别聚为一类,且与其他的基因明显分开。LDOX在处理1和处理2的果肉以及处理3的果心中的表达量与花色苷含量呈极显著相关,而F3GT在处理1的果肉和处理3的果心中的表达量与花色苷含量呈极显著相关。【结论】LDOX和F3GT可能是‘天源红’果实花色苷生物合成途径中的关键结构基因。套袋抑制LDOX在‘天源红’外果皮、果肉中的表达,而一直套袋促进其在果心中的表达。套袋抑制了F3GT在果肉中的表达,而对果心中F3GT的表达无明显影响。

[Objective] The aim of this survey is to explore the effect of bagging on expression of crucial structural genes of anthocyanin biosynthesis of different parts of kiwifruit fruits in different stages of growth and development and provide evidence for the impacts of illumination on anthocyanin synthesis. [Methods ] Full red-flesh fruits of kiwifruit ＇Tianyuanhong＇ were selected as materials for bagging treat- ments. According to the RNA-seq, 12 differential expression genes （DEGs） were screened. These 12 se- lected DEGs were conducted to analysis on the expression rule in the seven stages using quantitative real- time polymerase chain reaction （qRT-PCR）. Finally, the crucial candidate genes were confirmed based the results of qRT-PCR and cluster analysis. And then the relative quantitative analysis of expression level of the crucial candidate genes in different treatments, different periods and different parts of fruits was conducted by the qRT-PCR, and furthermore, the correlation analysis between expressions of genes and anthocyanin contents was also conducted. [Results]The expressions of 12 structural genes presented regular change rules although their expressions were different. The expression levels of F3H, LDOX and F3GT 110 days after full bloom （DAFB） when the flesh turned to be red were higher than those in other stages. The results of cluster analysis showed that LDOX and F3GT were respectively clustered into one class and were obviously separated from other genes. The expression level of LDOX of epicarp of treatment 1 dis- played an increase trend and reached peak on 110 d during the growth and development of fruits. While the levels of LDOX of epicarp of treatment 2 and treatment 3 were lower than that of treatment 1, especially on 100 d and 110 d. This indicated that bagging treatment and bagging-debagging treatment could decrease the expression level of LDOX in epicarp when the flesh turned to be red. The changing rule of LDOX expression level of sarcocarp of treatment 1 was similar to the trend of epicarp. As the developing of fruit, the expression level of LDOX increased and reached its maximum value on 110 d and it was evidently higher than those in other stages. While the expression level of expression LDOX of treatment 2 and treatment 3 was obviously lower than that of treatment 1. This phenomenon suggested that bagging treat- ment and bagging-debagging treatment could decrease the expression level of LDOX in sarcocarp. There was no obvious changing rule for the expression of LDOX of columella of treatment 1 and treatment 2. However, the expression level of LDOX of treatment 3 was higher than that of treatment 1 and tratment 2 on 110 d, which hinted that bagging treatment promoted the expression of LDOX in columella. The expression level of F3GT in epiearp of treatment 1, treatment 2 and treatment 3 displayed a similar changing rule during the period of growth and development of fruit. The expression level of F3GT increased from 90 d to 110 d, but the expression level of F3GT of treatment 1 was higher than that of treatment 2 and treat- ment 3, especially on 110 d. And the expression level of F3GT of treatment 2 was higher than that of treatment 3. These phenomenon indicated that bagging-debagging treatment could increase the level of F3GT in epiearp. The expression level of F3GT in sarcoearp of treatment l displayed an increase trend and reached peak on 100 d, which was the same as the expression level of LDOX in sarsocarp. The expressing of F3GT in eolumella of treatment 1, treatment 2 and treatment 3 had no obvious rule. The expression level of LDOX in sareoearp of treatment 1 and treatment 2 and the expression level of LDOX in columella of treatment 3 had a very significant correlation with anthoeyanin content. And the correlation between expression level of LDOX in epiearp and sarcocarp of treatment 3 and anthoeyanin content was statistically significant. The expression level of F3GT in sarcoearp of treatment 1 and the expression level of LDOX in eolumella of treatment 3 had a very significant correlation with anthocyanin content. And the correlation between expression level of LDOX in epicarp of treatment 3 and the expression level of LDOX in sarcocarp of treatment 2 and anthoeyanin content were also statistically significant. [Conclusion] LDOX and F3GT might be the crucial structural genes of anthoeyanin biosynthesis in the fruits of ＇ Tianyuanhong＇ ki-wifruit. Bagging treatment inhabited the expression of LDOX in epicarp and sareoearp, while it promoted the expression of LDOX in columella. Bagging-debagging treatment promoted the expression of F3GT in epicarp while it suppressed the expression of F3GT in sarsocarp, which was consistent with the expression rule of LDOX in sarcoearp. Bagging treatment had no obvious impact on expression of F3GT in eolumella.