Comparative Analysis Of β-carotene Hydroxylase Gene Between Peach And Apricot

With the continuous development of the economic level,consumers’ requirements for the appearance and nutritional value of the fruit have gradually increased.Carotenoids and their derived decarboxycarotenoids are important bioactive substances for forming fruit color and maintaining human health.Therefore,it is of great scientific significance and economic value to study the regulation of carotenoid metabolism in fruits.β-carotene hydroxylase is one of the key enzymes in the biosynthesis and metabolism of carotenoids in plants.It can catalyze the production of β-carotene into zeaxanthin.There was a significant difference in carotenoid composition between peach and apricot fruits with yellow flesh.The apricot fruits had a dominant accumulation of β-carotene,while the peach fruits had a dominant accumulation of lutein components downstream of β-carotene.Until now,the molecular mechanism that determines the difference in carotenoid composition between the two is unknown.This study took peach and apricot BCH genes as the research objective,and analyzed the gene expression and promoter activity to determine the role of BCH genes in determining the difference in carotenoid components in yellow-fleshed peach and apricot fruits.The main findings are as follows:1.The carotenoid content and accumulation characteristics of yellow peach and yellow apricot are compared and analyzed.The carotenoids detected in yellow peach and yellow apricot mainly include:β-carotene,P-cryptoxanthin,anther yellow In mature fruits,yellow apricots mainly accumulate β-carotene,and yellow peaches mainly accumulate violaxanthin.2.The BCH1 and BCH2 genes were cloned from yellow-fleshed peaches and apricots,encoding 311 and 295 amino acids,respectively.There are two amino acid differences in the BCH1 gene of peach and apricot.Sequence alignment showed that the sequence similarity of the two amino acids was about 99.35%,indicating that the BCH1 gene was relatively conservative,with very little variation in the evolutionary process.Phylogenetic tree analysis showed that both genes could be clustered with members of corresponding gene families with known functions,and both genes had high homology with Rosaceae fruit trees.3.Analysis of BCH gene expression.The expression levels of BCH gene in peach and apricot fruits at different maturity stages were analyzed by fluorescence quantitative PCR.The results showed that the expression level of BCH1 gene increased gradually with fruit development and maturity,and reached the highest at mature stage.Compared with fruits,BCH1 gene expression was lower in flowers,roots and leaves.There was no significant difference in the expression of BCH2 gene during fruit development.The expression level of BCH1 was significantly higher than that of BCH2,suggesting that BCH1 gene is a key gene affecting carotenoid synthesis.Especially in fruit,the expression level of BCH1 gene was significantly different between peach and apricot.4.Cloning of BCH1 gene promoter.Sequence analysis showed that the promoter region of BCH1 gene in peach and apricot was about 1400bp.,through the analysis of the effect on cis element BCH1 promoter exists in addition to the basic role in promoter cis structure element,there is light control components,hormone response,relevant parts of the organ specificity of components,reaction related components,such as low temperature response suggested BCH1 gene transcription by light,temperature,hormone,the influence of factors such as moisture and circadian rhythm.5.Analysis of transient expression in peach and apricot fruits.The peach and apricot fruits were injected with Agrobacterium tumefaciens containing PBI121-X and PBI121-T plant expression vectors,respectively,to analyze the expression of GUS gene in the peach and apricot flesh after injection.The results showed that the expression level of apricot flesh injected with PBI121-X vector was lower than that of apricot flesh injected with PBI121-T vector.The expression of GUS gene in apricot injected with PBI121-X and PBI121-T vectors was lower than that in peach injected with PBI121-T vectors,and there was no significant difference in the activity of the two vectors.6.The promoter activity of BCH1 gene was analyzed by transgenic tomato.Plant expression vectors pBI121-X and pBI121-T were constructed to drive GUS gene by BCH1 gene promoter in peach and apricot,respectively.Transgenic tomato lines were obtained by Agrobacteria-mediated method.GUS histochemical staining and GUS gene expression in transgenic tomato flesh were analyzed.The results showed that the activity of BCH1 gene promoter in peach and apricot increased with fruit development and ripening,but there was no significant difference between the two genes.