Study On Wax Structure, Wax Composition And The Key Wax Gene Expression During The Cold Storage Of ’Gan Qi-3’

This paper aims to compare ?Gan Qi- 3‘ Navel Orange with Newhall navel orange at low temperature storage: wax structure, the total wax quality, wax composition differences; and Transcriptome sequencing(RNA-Seq) was performed to examine differentially expressed genes;quantitative real-time PCR were used to confirm their expression levels. These studies will lay a theoretical foundation for improving the quality of navel orange. The main results are as following:1.with the cold storage, vertical plate and tubular wax crystal number is increased of ?Newhall‘ navel orange, and ‘Gan Qi- 3’ Navel Orange increased tubular wax crystal number. The waxes of citrus fruit surfaces were mostly composed of Saturated fatty acids, unsaturated fatty acids, alkanes, primary alcohols and triterpenoids small-molecular metabolites, waxy structure、component proportion and contents all have differences between ?Newhall‘ navel orange and ‘Gan Qi- 3’ Navel Orange.2. Glossy navel orange and ‘Gan Qi- 3’ Navel Orange were sequenced by Illumina HiSeqTM 2000, results of RNA-Seq appear below:(1)For ‘Gan Qi- 3’ Navel Orange, before the storage,12,081,622 high-quality reads were generated;After storage for 90 days,11,795,487 high-quality reads were generated;After storage for 150 days,11,885,027 high-quality reads were generated。For Newhall navel orange, before the storage,11,590,101 high-quality reads were generated;After storage for 90 days,12,081,005 high-quality reads were generated;After storage for 150 days,12,340,256 high-quality reads were generated. The same variety but different temperature storage period and the same temperature storage but different species have different gene coverage.(2)We find 24 passway between ADZ and ABY, 56 passway between BDZ and BBY, 44 passway between CDZ and CBY, 118 passway between ABY and BBY, 124 passway between ABY and CBY, 118 passway between BBY and CBY, 105 passway between ADZ and BDZ, 123 passway between ADZ and CDZ, 99 passway between BDZ and CDZ.Overview of group differentially expressed genes(probability≥0.8 AND |log2Ratio|≥1), 597 genes were found to be up-regulated and 510 genes were down regulated of BBY vs ABY; 1751 genes were found to be up-regulated and 843 genes were down regulated of CBY vs ABY; 26 genes were found to be up-regulated and 13 genes were down regulated of ABY vs ADZ; 494 genes were found to be up-regulated and 280 genes were down regulated of BDZ vs ADZ; 1591 genes were found to be up-regulated and 484 genes were down regulated of CDZ vs ADZ;878 genes were found to be up-regulated and 397 genes were down regulated of CBY vs BBY; 70 genes were found to be up-regulated and 99 genes were down regulated of BBY vs BDZ; 321 genes were found to be up-regulated and 256 genes were down regulated of CDZ vs BDZ; 22 genes were found to be up-regulated and 87 genes were down regulated of CBY vs CDZ.(3) Differentially expressed genes were categorized into three main categories by gene ontology(GO): biological process, cellular component, and molecular function.(4) The pathway by KEGG were involved the most in metabolic pathway, and were involved the second in the biosynthesis of secondary metabolites.3.Combined RNA-Seq data with waxy different composition results, differentially expressed genes related to wax synthesis were selected from KEGG pathway. To understand the formation mechanism of ‘Gan Qi- 3’ Navel Orange at gene level, RT-qPCR were used to examine the expression pattern during navel orange fruit development. Main results are as follows:(1) 15 differentially expressed genes in pathway were choosen randomly to demonstrate the RNA-seq results. 6 were down-regulated and 9 were up-regulated among them. 14 genes expression were continual while the rest performed a discontinuity. The TR-qPCR data were basically consistent with the RNA-seq.(2) Maybe the key passway of causeing different storage performance between ‘Gan Qi- 3’ Navel Orange and Newhall navel orange is Cutin, suberine and wax biosynthesis、Fatty acid biosynthesis、Biosynthesis of unsaturated fatty acids、ABC transporters 、Fatty acid elongation、Phenylpropanoid biosynthesis.(3) During the low temperature storage,CsABCG2-1,CsCYP76M7,CsREF1,CsFAR,CsKCS,CsCER1 down regulated gene is invole in wax biosynthesis and transporters of ‘Gan Qi- 3’ Navel Orange peel. Those gene have the same performance with wax composition such as alkanes, primary alcohols.