Cloning Of MiPAO And MiRCCR Genes And Their Roles In Chlorophyll Degradation Of Mango Peel

Mangifera indica L.as evergreen fruit tree,belongs to the Anacardiaceae genus Mandicaceae,is one of the five famous tropical fruits,distributed mostly in the tropics and subtropics in the world.The color of the mango fruit when its maturation directly influences people's affection,thus determines the commercial value of the mango.The chlorophyll degradation pathway affects the formation of fruit color.Three different coloring types(red'Guifei';yellow'Jinhuang';green'Guiqi')of mango peel as materials,the MiPAO and MiRCCR genes were cloned,bioinformatics analyzed,expression analyzed,and physiological indexes related to fruit coloring were determined.The construction of virus-induced gene silencing and over-expression vectors of plant in Micro-Tom tomato was used as transgenic plants research.The role of MiPAO and MiRCCR genes was clarified in the chlorophyll degradation of pathway.The main results as follows:1.The MiPAO and MiRCCR genes were cloned successfully,and bioinformatics analyzed.The full-length cDNA sequences of PAO and RCCR genes were got by 3 'and 5'rapid-amplification of cDNA ends(RACE)technique in mango pericarp from three different coloring mango varieties,named MiPAO and MiRCCR by bioinformatics analysis.The results showed that the cDNA sequences of the MiPAO and MiRCCR genes in the three coloring varieties were differed by only a few bases.For example,the full-length cDNA sequence of the MiPAO gene in the red 'Guifei' mango fruit was 1,979bp,and the open reading frame was 1,641 bp,encoded 546 amino acids,with a molecular weight of 61.816 kD,an isoelectric point of 6.54.As an example,the full-length cDNA sequence of the MiRCCR gene in the yellow 'Jinhuang' mango was 1,134 bp,the open reading frame was 975 bp,and encoded 324 amino acids,the molecular weight was 36.21 kD,and the isoelectric point 5.13.Three tertiary protein structures of the MiPAO and MiRCCR genes were predicted by biological software.A phylogenetic tree was constructed using the sequences of 30 different plants which registered on the NCBI.It was found that the protein encoded by the MiPAO gene was closely related to fruit trees such as oranges,and the protein encoded by the MiRCCR gene is more related to fruit trees such as oranges,and citrus clementine.2.The physiological indexes related to pericarp coloring of three different mango varieties were determined.The content of chlorophyll was determined in soft-ripe stage by spectrophotometry.The results showed that the contents of chlorophyll a,chlorophyll b and total chlorophyll in green 'Guiqi' mango,were significantly higher than those in red'Guifei' and yellow 'Jinhuang',the content of chlorophyll was the lowest in yellow'Jinhuang'.The main components of anthocyanins and the content of ?-carotene in mango peel were determined by HPLC.The results showed that Delphinidin-3-O-galactoside was the major components of anthocyanin in red 'Guifei' mango and yellow 'Jinhuang' mango.Malvidin-3-O-galactoside is the main component of anthocyanin in green 'Guiqi' mango.The content of ?-carotene in red 'Guifei' mango was 4.6 times as much as that in yellow'Jinhuang' mango and 9.1 times as much as green 'Guiqi'.The results showed that the formation of peel color in 'Guifei' and'Jinhuang'mango was closely related to the accumulation of ?-carotene.3.The expression levels of MiPAO and MiRCCR genes in the pericarp of three cultivars were analyzed.The expression of MiPAO and MiRCCR genes in the peels of three varieties of soft-ripening period were detected by qRT-PCR.The results showed that the relative expression levels of MiPAO and MiRCCR genes were the highest in the green'Guiqi',followed by the red 'Guifei',and the lowest in the yellow 'Jinhuang'.4.Construction of virus-induced gene silencing vector and plant overexpression vector in Micro-Tom tomato was used as the functional researchof transgenic plants.The pTRV2-MiPAO and pTRV2-MiRCCR gene silencing vectors and pGreen? 62-SK-MiPAO and pGreen? 62-SK-MiRCCR over-expression vectors were successfully constructed by using the cloned MiPAO and MiRCCR gene sequences.Fruit stem injection after a week of flowering and peel surface injection at green fruit stage were used,transformation of Micro-Tom Tomato by agrobacterium tumefaciens mediated injection.The results showed that fruit stem injection was much more intuitive and more effective than the phenotype of the fruit surface in the phenotype of the Micro-Tom tomato.Through the observation of Micro-Tom tomato phenotype,determination of chlorophyll,carotenoid content,and semi-quantitative PCR detection,the silencing of SIPAO and SIRCCR genes in Micro-Tom tomato inhibited chlorophyll degradation near the injection site,resulting in fruit color differences and partial slow green,at the same time,the synthesis and accumulation of carotenoids are also inhibited.On the other hand,The over-expression of MiPAO and MiRCCR.in Micro-Tom tomato could accelerate the color conversion,and showed the character of "prematurity".These results provides some experimental support for fruit color formation and color regulation during the maturation of mango fruit,and it also provides a theoretical basis for controlling the color of the mango fruit and regulation the ripening period.