Preliminary Study On The Function Of CmABCB19 And CmABCC10 In Fruit Shaping Of Fingered Citron

Citri sarcodactylis fructus is derived from the fruit of Citrus medica L.var.sarcodactylis Swingle.It is used as medicine after the immature fruit is thinly sliced and dried.The wide end is finger-like split,and the edge epidermis is green-brown or yellow-brown.The one with intact pieces,green skin and white meat,and delicate fragrance is the best.The characteristics of medicinal materials are directly related to the development of fresh fruit.In the course of the general survey,we found that the shape of fruits in some trees is different.In addition to the common fruit with finger-like closure at the top,there are fruit similar to citrus in shape,so we named them ’normal fruit’ and’malformed fruit’.This study evaluated the appearance quality of two kinds of fruits,and identified the key genes regulating the formation of malformed fruits through transcriptome sequencing and gene family analysis.In the Saccharomyces cerevisiae genetic transformation system,transient expression system of tobacco and Arabidopsis thaliana genetic transformation system,we verified the function of the key genes,and initially revealed the reason of malformed fruit formation.The main results are as follows:1)To evaluate the apparent quality of normal and malformed fruit,including fruit size,peel color and pigment content.The normal fruit is oval,with finger-like lobes,and the fruit shape index is greater than 1.2;The malformed fruit is flat,and the fruit shape index is 0.4-0.6.The brightness value L*and yellowing value b*of malformed fruit are higher than the normal,and the greenish value a*is lower than normal fruit.The content of total chlorophyll and carotenoid in malformed fruit was lower than the normal,but the ratio of total chlorophyll and carotenoid content was significantly higher.2)Identified the key genes regulating the formation of malformed fruit.The transcriptome analysis of the normal and malformed fruits of C.medica showed that the expression of DEGs in the ABC transporter pathway of the malformed fruit was down regulated.According to the sequence of ABC transporters in Arabidopsis,we identified 119 members of ABC transporter gene family in C.medica.The key transporter genes CmABCB19 and CmABCC10,were obtained through comprehensive screening and were successfully cloned.The expression of transporters CmABCB19 and CmABCC10 in the normal fruit was higher than that in the malformed fruit,which was consistent with the transcriptome.Then,we compared the target gene with the conserved domain of ABC transporter of other species,and further speculate that CmABCB19 transporter could transport auxin IAA,and CmABCC10 transporter could transport flavonoids.3)The content of auxin and flavonoid in the normal and malformed fruits of C.medica was determined.The content of auxin in fruits was analyzed by UPLC-3QMS.The results showed that the content of IAA and IB A in the normal fruit was higher than that in the malformed.Using UPLC-Q-TOF-MS to qualitatively analyze the flavonoid components,13 kinds of flavonoid components were identified,and further analysis showed that the contents of naringin,diosmin and nobiletin in the malformed fruit were higher than those in the normal fruit,while the contents of rutin and hesperidin were lower.4)The gene transformation system of S.cerevisiae was used to verify the function of the transporter.The wild type,mutant and complementary mutant strains of S.cerevisiae were incubated in the culture medium with substrate,and the content of accumulated substrate in the yeast cell was measured at the same time.It was speculated that CmABCB19 could transporter auxin in yeast cells,CmABCC10 could make naringin,citrinin and vanillin turn in,and make rutin and hesperidin turn out.5)Using the tobacco transient transformation system to observe subcellular localization,the results indicated that the CmABCB19 was localized in the cell membrane and the CmABCC10 was localized in the vacuolar membrane.Using the genetic transformation system of A.thaliana to verify the function,compared with the mutants,the gene expression in auxin biosynthesis pathway and signal transduction pathway in abcb19-CmABCB19 was up-regulated,and the content of auxin was increased,while the content of rutin and naringin was increased.CmABCB19 in C.medica could participate in auxin transport and affect the pathway of flavonoids synthesis and accumulation.Compared with the mutants abcc10,the expression of flavonoid synthesis pathway gene in abcc10-CmABCC10 was up-regulated,the content of rutin and naringin increased,and the content of auxin increased.CmABCC10 can participate in the transport of flavonoids and regulate the synthesis and transport of auxin.Meanwhile,CmABCB19 and CmABCC10 also have an impact on the development of pods and seeds in Arabidopsis.In summary,we speculate that the expression level of ABC transporters in malformed fruits is inhibited,resulting in differences in the content of auxin and flavonoids in the fruits,which in turn leads to changes in the apparent and intrinsic quality of the fruits.