| In this study,transgenic Arabidopsis overexpressied CsSHN1(a waxrelated transcription factor of navel orange)and wild-type Arabidopsis were used as materials.To analyze the function of CsSHN1 in controlling plant wax biosynthesis,gas chromatography-mass spectrometry(GC-MS)was used to compare the total wax and cutin amounts in leaves and stems of transgenic Arabidopsis,and wild-type Arabidopsis.The wax and cutin composition in leaves and stems were also compared between the two kind of Arabidopsis.At the same time,transcriptome sequencing for stems,leaves and flowers of two kind of Arabidopsis was carried out to screen the up-regulatied and down-regulatied wax synthesis and export genes caused by overexpression of CsSHN1,so as to explore the downstream wax genes regulated by CsSHN1 transcription factors.Transcriptome sequencing results were verified by RT-qPCR.The promoter sequences of CsSHN1 gene were cloned from "Newhall navel orange" and "Ganqi 3".The functional elements were analyzed by PLACE promoter on-line analysis program.The expression vector for CsSHN1 promoter activity detection was constructed,and transformed into Arabidopsis.Homozygous transgenic plants were was used to detect the promoter activity by GUS histochemical staining.The result of promoter activity detection will provide foundation for exploring upstream regulatory genes interacting with CsSHN1 promoter.The main findings are as follows:1.GC-MS results showed that the total amounts of wax and cutin in CsSHN1 overexpressied Arabidopsis stems were significantly higher than that in wild type Arabidopsis stems.The amounts of saturated fatty acids,alkanes,aldehydes,primary alcohols and steroids fractions were also significantly higher than those stems in wild type Arabidopsis.The contents of unsaturated fatty acids and triterpenoids were not significantly different between the stems of two kinds of Arabidopsis.The content of dicarboxylic acids in cutin of transgenic Arabidopsis stems was significantly higher than that in wild type Arabidopsis.The total amount of wax and cutin in transgenic Arabidopsis leaves were also significantly higher than that of wild type Arabidopsis.The amounts of fatty acids,alkanes and steroids in transgenic Arabidopsis leaves were also significantly higher than those in wild type Arabidopsis leaves.The middle straight chain fatty acids,the terminal hydroxyl straight chain fatty acids and the straight chain dicarboxylic acids in transgenic Arabidopsis leaves were significantly higher than wild type Arabidopsis.2.The sequencing results based on the Illumina HiSeq X-ten high-throughput sequencing platform are as follows:(1)For wild-type Arabidopsis,50253016 high-quality reads were generated in the stems,50476328 high-quality reads were generated in the flowers,53789488 high-quality reads were generated in the leaves;For transgenic Arabidopsis,50725828 high-quality reads were generated in the stems,54490794 high-quality reads were generated in the flowers,51860240 high-quality reads were generated in the leaves.(2)4634 differentially expressed genes(DEGs)were identified in all samples.In the WTS-VS-MTS comparison group,2302 DEGs were detected,including 1514 up-regulated genes and 788 down-regulated genes.In the WTF-VS-MTF comparison group,there were591 DEGs,including 343 up-regulated genes and 248 down-regulated genes.In the WTS-VS-MTS comparison group,1741 DEGs were detected,including 1251 up-regulated genes and 490 down-regulated genes.(3)DEGs were enriched into three main categories by gene ontology(GO): cellular component,molecular function,and biological process.Most DEGs were enriched in nucleus,plasma membrane,extracellular region,sequence-specific DNA binding transcription factor activity and transcriptional regulation of transcriptionand DNA-templated,which respectively accounted for 34.2%,16.7%,15.0%,14.2%,11% of the total DEGs.(4)The KEGG pathway enrichment analysis of DEGs showed that there were 106 different pathways in WTS-VS-MTS comparison group,among which anthocyanin biosynthesis pathway was the most significant pathway;And 63 differentially expressed pathway in WTF-VS-MTF comparison group,the photosynthesis-antenna protein pathway was the most significant pathway.100 different pathways were enriched in WTL-VS-MTL comparison group,in which cutin,suberine and wax biosynthesis pathway is the most significant pathway.(5)Seven different pathways related to wax synthesis and transport were screened in WTS-VS-MTS comparison group,which were cutin,suberine and wax biosynthesis,terpenoid backbone biosynthesis,fatty acid biosynthesis,fatty acid extension and fatty acid metabolism,ABC-transporter pathway,sesquiterpenoid and triterpenoid synthesis pathway.49 DEGs were found in these pathways,of which 44 genes were up-regulated and 5 genes were down-regulated.Five differential pathways related to wax synthesis and transport were screened in WTF-VS-MTF comparison group,which were cutin,suberine and wax biosynthesis,terpenoid backbone biosynthesis,fatty acid biosynthesis,fatty acid extension and fatty acid metabolism.16 differentially expressed genes were found in these pathways,including 10 up-regulated genes and 6 down-regulated genes.Seven differential pathways related to wax synthesis and transport were screened in WTL-VS-MTL comparison group,which were cutin,suberine and wax biosynthesis,terpenoid backbone biosynthesis,fatty acid biosynthesis,fatty acid extension and fatty acid metabolism,ABC-transporter pathway,sesquiterpenoid and triterpenoid synthesis pathway.36 DEGs were found,of which 28 genes were up-regulated and 8 genes were down-regulated.3.According to the results of transcriptome sequencing,21 wax-related DGEs were randomly selected for RT-qPCR validation.The results showed that 16 genes were up-regulated and 5 genes were down-regulated.The results were consistent with the results of transcriptome sequencing,which indicated that the results of sequencing were reliable.4.Combined with GC-MS results,transcriptome sequencing results and RT-qPCR validation results,we screened CER1,CER4,KCS family genes,FAR family genes,LACS family genes,ABCB family genes,LUP family genes as the candidate genes for the downstream genes controlled by CsSHN1 transcription factors.5.The promoter sequence of CsSHN1 gene was cloned from “Newhall navel orange”and “Gan navel 3”,and these two sequences were completely identical.The functional elements in CsSHN1 promoter sequence were analyzed by PLACE promoter on-line analysis program.It was found that the sequence contained lighting-response elements,plant hormone response elements,abiotic and biotic stress response elements,which suggested that CsSHN1 might be related to citrus stress response.6.The expression vector for CsSHN1 promoter activity detection was constructed,then transformed to Arabidopsis and obtained homozygous transgenic plants.The results of GUS histochemical staining for detecting CsSHN1 promoter activity showed that blue color in the leaves,stems and flowers of transgenic Arabidopsis.It suggested CsSHN1 promoter could induce the expression of downstream gene GUS,and had the highest activity in flowers. |
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