Effect Of Avoiding Light On Bitter And Astringent Substances Content And Expression Of Key Biosynthetic Genes In Dendrocalamus Latiflorus

Ma bamboo(Dendrocalamus latiflorus Munro)is one of the most important bamboos in China.Ma bamboo shoot is a high-protein,high-fiber,low-sugar and low-fat green health food,which contains a lot of minerals,vitamins and trace elements.However,they will taste much more bitter and astringent once unearthed and exposed to the sunlight,which seriously restricts the development of bamboo shoot industry.In this experiment,bamboo shoots of natural growth(CK)and earth-covered(EP)were used as experimental materials.The bitter and astringent substances in the two types of bamboo shoots were determined by high performance liquid chromatography,spectrophotometry,bromocresol green acid dye method and isonicotinic acid-pyrazolone colorimetric method.The correlation between substance content and bitterness intensity of bamboo shoots was analyzed.Simultaneously,the transcriptomes of two bamboo shoots were sequenced by the Illumina Hi Seq TM 2500 platform and the differentially expressed genes in response to light protection were screened.The results are as follows:1.Determination of bitter and astringent substances and their correlations with bitterness and astringency in Ma bamboo shoots.(1)The contents of bitter amino acids(Phe,Tyr,Leu),cyanide,linamarin,lotaustraline,oxalic acid,flavonoid,alkaloids,tannin in Ma bamboo shoots under earth-covering conditions were lower than natural Ma bamboo shoots,and the contents of flavor amino acids(Asp?Glu)were more than those of natural Ma bamboo shoots,and the contents of each substance in two types of bamboo shoots were different in significant level.(2)The bitterness and astringency of the Ma bamboo shoots were positively correlated with the contents of Leu,linamarin,oxalic acid and alkaloids,and they were significantly positively correlated with the content of Tyr,Phe,cyanide,lotaustraline,tannin and flavonoids,and they were significantly negatively correlated with Asp and Glu.2.Transcriptome analysis results.(1)By the means of transcriptome sequencing,36.45 Gb raw data of 6 bamboo shoot samples were obtained from CK and EP,and 53388 Unigenes were assembled.The number of Unigenes annotated in Nr,GO,KEGG,Swiss-prot,Pfam,and COG databases were 30,979(58.03%),24925(46.69%),6294(11.79%),21,245(39.79%),16346(30.62%)and 9875(18.50%)respectively.The number of total annotated Unigenes was 31,462(58.93%),and the species with the highest proportion of homologous sequences was rice.A total of 8808 SSR sites were found in the spliced Unigenes,which were distributed in 6,366 Unigenes.The frequency of appearance was 16.50% and the frequency of occurrence was 12.43%.The SSR repeat type of largest number was three-base repeat,accounted for 41.13% of the total number of SSR.(2)A comparative analysis of the expression levels of Unigene detected by CK and EP resulted in the screening of 1846 differentially expressed genes,of which 998 were up-regulated and 848 were down-regulated in EP.The differentially expressed genes were aligned to the GO database and a total of 701 genes were annotated.The GO classification statistics showed that the differentially expressed genes accounted for a large proportion of degradation processes and sub-categories of enzyme activity.The differential genes yielded a total of 235 functional definitions when annotated in the KEGG database,with 32 significantly enriched pathways.The metabolic pathways involved in the synthesis of bitter and astringent substances in Ma bamboo shoots included glycolysis/gluconeogenesis,phenylalanine,tyrosine and tryptophan biosynthesis,phenylpropane biosynthesis,alanine,pyruvate metabolism,etc.(3)According to the function definition of Unigene in each database,combined with the enrichment pathway of differential genes in KEGG database,the related genes of bitter and astringent substances in Ma bamboo shoots were searched and analyzed.It was found that the expression levels of phenylalanine,tyrosine,and key tannin biosynthetic enzymes PFK,ENO,HPP-AT/PPY-AT,and LAR were significantly reduced under light-proof conditions.It may eventually affect the synthesis of phenylalanine,tyrosine,and tannin in the Ma bamboo shoots,which in turn reduces the bitter astringency of the bamboo shoots.3.Real-time fluorescence quantitative reverse transcription PCR.Four genes of PFK,ENO,HPP-AT/PPY-AT,and LAR which related to the synthesis of tannin and bitter-tasting amino acids were selected for RT-q PCR validation.The results showed that the expressions of selected genes in EP treatment were lower than those of CK,and there was a significant difference at the level of P?0.01.In this experiment,the effect of light on the synthesis of bitter and astringent substances in Ma bamboo shoots was preliminary explored from physiological and molecular levels.It provides a reference for further understanding of the light-affecting mechanism of the synthesis of bitter and astringent substances,and also lays a theoretical foundation for future research on cloning and functional analysis of related genes.