基于转录组研究光质对转色期红地球葡萄果实着色及品质的影响

Effects of light quality on the berry coloration and quality of Red Globe grape during veraison based on transcriptome sequencing

【目的】探讨不同光质对设施红地球葡萄果实着色及品质的影响,并从中挖掘出关键的光调控基因。【方法】以8年生红地球葡萄为试验材料,采用4种不同光质(红蓝光2:1、蓝光、红光、白光)补光处理设施红地球葡萄,以不补光为对照,对果实中花青素、还原糖、可滴定酸和可溶性固形物含量进行测定,并对果皮进行转录组测序。【结果】不同光质处理下果实中花青素、还原糖和可溶性固形物含量均高于对照,其中在蓝光处理下最高。利用FPKM计算基因表达量,以差异表达倍数|log2fold changes|≥1,P-adjust<0.05为筛选条件,共获得1313个差异表达基因,包括上调基因728个,下调基因585个,其中红蓝光、蓝光、红光、白光处理与对照之间的差异基因数目分别为638、434、375和320个。GO分析发现差异基因与代谢过程、细胞过程、膜、催化活性、结合等条目密切相关。KEGG分析发现不同光质通过调控葡萄光合作用、类黄酮生物合成、淀粉和蔗糖代谢等相关基因表达进而影响葡萄果实着色及品质。根据富集结果选取9个差异表达基因,经qRT-PCR验证,基因表达趋势与转录组测序结果一致。【结论】不同光质补光均能改善葡萄果实的着色及品质,其中以蓝光的效果最为明显;葡萄果实中类黄酮基因CHS、F3H及淀粉和蔗糖代谢基因INV、SPS、HK和BG的差异表达,是导致各处理葡萄果实着色及品质差异的重要原因。

【Objective】Greenhouse grape has the characteristics of early berry bearing and high benefit,which has developed rapidly in Ningxia and significantly improved the economic benefit of local grape industry.Red Globe grape is the main variety of table grape in Ningxia,which has the characteristics of big ear,brilliant color,sweet taste,storage tolerance and high yield,etc.However,under the condition of protected cultivation,weak light and poor light quality will affect the color set and maturity of grapes,resulting in the decline of grape yield and quality,which restricts the sustainable development of protected grape industry.As the main environmental factor for the growth and development of protected horticultural crops,light not only affects the photosynthetic capacity of protected horticultural crops,but also participates in the regulation of various physiological processes.As an effective measure to improve lighting conditions in facilities,LED light supplement can accurately regulate spectral composition,and is widely used in facility cultivation research.Transcriptome sequencing has been applied in the study on gene structure,function and expression,it is also an important method for phenotypic correlation research,which plays an important role in exploring the molecular mechanism of plant,parsing facility grape berry coloring and quality influencing factors,and helping to improve the grape commodity value,so as to improve the economic benefits of grape planting.【Methods】The experiment was carried out in the shade of the solar greenhouse from April to September,2019.The eight-year-old Red Globe grapes were used as experimental materials,and four different kinds of light quality treatments were set up in this experiment.They were red blue light(red light:blue light=2:1),blue light,red light and white light,with no supplementary light as the control.Twelve vines with the same growth vigor were selected for each treatment and divided into 3 replicates with 4 vines in each replicate.Supplementary light lamps were placed 30 cm above the tree in the test row,and 6 supplementary light lamps were set for each treatment.All treatments were separated by reflective film.Lightening started from April 2019,from 8:00 to 20:00 every day,with a light intensity of 200μmol·m-2·s-1,until the end of berry ripening and harvesting.The net photosynthetic rate(Pn),stomatal conductance(Gs),intercellular CO2 concentration(Ci)and transpiration rate(Tr)of grape leaves were measured at the berry-color-turning stage.The contents of anthocyanins,reducing sugars,titratable acids and soluble solids in the berry were determined,and transcriptome sequencing was performed on the pericarp.The gene expression was calculated using FPKM and the screening criteria were differential expression multiple|log2fold changes|≥1 and p-adjust<0.05.The differentially expressed genes in the gene collection were compared with GO and KEGG databases to obtain the functional annotations and related metabolic pathway information of the genes in different samples.In order to verify the accuracy of RNA-seq data,9 differentially expressed genes were randomly selected for real-time fluorescence quantitative expression verification analysis.【Results】Red light increased the net photosynthetic rate and transpiration rate of leaves,while blue light increased stomatal opening and intercellular CO2 concentration.Different light quality had different effects on the photosynthetic performance of Red Globe grape leaves,and supplemental light could promote the photosynthesis of leaves.Blue light treatment had the best effect on improving berry color and quality,followed by red blue light,white light and red light.A total of 1313 differentially expressed genes were obtained in all the samples,including 728 up-regulated genes and 585 down-regulated genes,among which 638,434,375 and 320 differentially expressed genes were detected between red and blue light,blue light,red light,white light treatment and control,respectively.GO analysis showed that differential genes were closely related to metabolic process,cellular process,membrane,catalytic activity,binding and other items.Enrichment analysis of KEGG pathway found that the differential genes were mainly involved in photosynthesis,flavonoid biosynthesis and starch and sucrose metabolism pathways.Different light quality affected grape berry coloration and quality by regulating the expression of related genes such as grape photosynthesis,flavonoid biosynthesis,and starch and sucrose metabolism.Nine differentially expressed genes were randomly selected according to the enrichment results,and the gene expression trend was consistent with the results of transcriptome sequencing by qRTPCR.【Conclusion】Different light quality affected plant photosynthesis,further influencing the synthesis of organic matter such as sugar and amino acid.Light quality regulated the expression of genes related to the process of anthocyanin metabolism and the synthesis of anthocyanin.Blue light and white light significantly promoted the biosynthesis of anthocyanin in berry.Different light quality promoted the accumulation and conversion of starch and sucrose in berry,and improved berry quality.The up-regulated expression of INV,SPS,HK and BG genes in berry accelerated the sucrose metabolism and increased the accumulation of reducing sugars,while reducing sugars and soluble solids may have a promoting effect on the synthesis and accumulation of anthocyanins.Trehalose related genes were significantly up-regulated in white light,blue light and red blue light,suggesting that trehalose related genes played an important role in grape environmental adaptability.In addition,it was also found that supplementing light affected the metabolism of some amino acids and the expression of endogenous hormonerelated genes in berry,which may also be the important factors causing the differences in color and quality of grapes among different treatments.