Studies On The Different Mechanism Of Ethylene Release In Fruits Of Nanguo Pear And Its High Sucrose Accumulated Bud Sport

Nanguo pear is a typical climacteric fruit.The ripening of Nanguo fruit is mainly controlled by the plant hormone ethylene.The study of ethylene synthesis during fruit ripening is great significance to the artificial control of fruit storage and preservation.Ethylene synthesis is affected by many factors.In addition to temperature,light,plant hormones and other factors,ethylene synthesis can be regulated.Recent studies have shown that exogenous sucrose treatment can also regulate ethylene synthesis in fruits,but the molecular mechanism of this regulation is still unclear.In this study,Nanguo pear and the bud sport of its fruits were used as test materials.We measured the ethylene content in the fruits of two varieties and found that the peak of ethylene release was earlier in the bud sport of Nanguo fruits.and it was speculated that the peak of ethylene release was caused by sucrose accumulation.Ethylene synthesis genes Pu ACS1 a and Pu ACO1 and the transcription factor Pu WRKY31 gene highly expressed in Nanguo and its bud sport of its fruits were mined through transcriptome sequencing.Further studies showed that high expression of Pu WRKY31 could lead to an earlier ethylene release peak and earlier fruit ripening by transcriptional regulation binding to the promoter of Pu ACS1 a and Pu ACO1,the key genes in ethylene synthesis,and promoting their expression.while the expression of Pu WRKY31 and ethylene release were increased in Nanguo pear fruits treated with exogenous sucrose.The main results are as follows:1.The peak of ethylene release in Nanguo pear fruit with bud sport was earlier than Nanguo pear fruit.It was found that the peak of ethylene release occurred in the bud sport fruits of Nanguo pear on the 9th day after harvest,while the peak of ethylene release occurred in the fruits of Nanguo pear on the 12 th day after harvest by measuring the ethylene production during muture.The expression peaks of Pu ACS1 a and Pu ACO1,the key genes of ethylene synthesis,appeared earlier in Nanguo pear fruits than in its bud spot fruits.The transcriptome sequencing results of Nanguo pear and its buds sport fruits at the harvest stage were analyzed,and the key genes of ethylene synthesis,Pu ACS1 a and Pu ACO1,which were highly expressed in bud sport of Nanguo pear were excavated.The expression levels of Pu ACS1 a and Pu ACO1 in bud sport fruits were significantly higher than those in Nanguo pear fruits at0 d and 5 d postharvest by q RT-PCR,and were significantly lower than those in Nanguo pear fruits at 10 d and 15 d postharvest by q RT-PCR.Sequences of the coding region and promoter region of this gene were cloned between the two cultivars,and no difference was found.2.The expression peaks of Pu WRKY31 appeared earlier in Nanguo pear fruits than in its bud spot fruits.It was found WRKY transcription factor binding domain by analyzing Pu ACS1 a and Pu ACO1 promoter sequences,and found that the most significant difference expression of transcription factor Pu WRKY31 by the transcriptome data.q RT-PCR analysis showed that the expression level of Pu WRKY31 in bud sport fruits was significantly higher than Nanguo pear fruits at 0 d and 5 d after harvest,and was significantly lower than Nanguo pear fruits at 10 d and 15 d after harvest.The expression trend was the same as that of Pu ACS1 a and Pu ACO1.3.Overexpression of Pu WRKY31 promoted the expression of Pu ACS1 a and Pu ACO1 and ethylene production in Nanguo pear fruits.The overexpression of Pu WRKY31 in Nanguo pear fruits induced the expression of key genes of ethylene synthesis and promoted the synthesis of ethylene in fruits.Silencing Pu WRKY31 inhibited the expression of Pu ACS1 a and Pu ACO1 and the production of ethylene in the bud sport fruits of Nanguo pear.These results indicated that Pu WRKY31 was a positive regulator of ethylene synthesis in Nanguo pear fruits.4.The Pu WRKY31 transcription factor binds to the Pu ACS1 a and Pu ACO1 promoters and upregulates their expression.Pu WRKY31 was found to bind the promoters of the key genes of ethylene synthesis,Pu ACS1 a and Pu ACO1,in vitro by Y1 H.Ch IP-PCR assay was used to confirm that Pu WRKY31 could bind the promoters of Pu ACS1 a and Pu ACO1 in vivo.GUS reporter assay showed that Pu WRKY31 promoted the transcription of Pu ACS1 a and Pu ACO1.5.Exogenous sucrose treatment increased the expression of Pu WRKY31,Pu ACS1 a and Pu ACO1 in Nanguo fruits,and promoted the ethylene synthesis of fruits.In order to verify that exogenous sucrose can promote ethylene synthesis in fruits,the fruits of Nanguo pear on the tree were treated with 0.3% sucrose and 0.3% mannitol as control.After 5 d,the fruits were harvested,and the ethylene production and related gene expression levels were measured.It was found that sucrose treatment up-regulated the expression of Pu WRKY31 in fruits,and then induced the expression of Pu ACS1 a and Pu ACO1,which promoted ethylene synthesis.In order to verify that endogenous sucrose promotes ethylene synthesis in fruits,agrobacterium mediated transient expression system was used to overexpress sucrose transporter Pu SWEET15 in the fruits of Nanguo fruits on the tree,and it was found that ethylene production in fruits were significantly increased,and the expression of key genes of ethylene synthesis was promoted.The silencing of Pu SWEET15 in bud sport fruits on trees showed a significant decrease in sucrose content,ethylene production and the expression of key genes in ethylene synthesis were inhibited.These results indicate that sucrose promotes ethylene synthesis in fruits.These results suggested that sucrose could promote the expression of Pu WRKY31 in bud sport fruits,and the highly expressed Pu WRKY31 could bind to the promoters of Pu ACS1 a and Pu ACO1,the key genes of ethylene synthesis,and increase their promoter activity,promote ethylene production,leading to an earlier ethylene release peak and earlier fruit ripening.This study provided theoretical support for fruit ripening and breeding of respiratory trophy.