The Cloning And Expression Analysis Of Key Genes Of Gibberenllin Metabolism Pathway In Loquat

Loquat?Eriobotrya japonica?belongs to Rosaceae maloideae Eriobotrya plants,native to China.Its fruits usually have plenty of relatively large seeds which accounts for about 20%-30% of the total weight of the fruit.Each fruit contains 3-5seeds.Thus,seedless traits become one of the most important breeding targets of loquat fruit.Gibberellins?GAs?is one of the important hormones,not only involved in plant growth regulation,but also can promot the fruitset of loquat.In this study,we adopt the diploid of Dawuxing and its triploid lines as the materials,and ten kinds of plant hormones were determined in the fruit development.Five key enzyme genes including GA20ox1,GA20ox2,GA3 ox,GA2ox,CYP714 A and CYP714 C downstream of loquat gibberellin metabolic pathways were cloned by homologous method and analyzed the expression of the key enzyme genes in early development.We aim at exploring the mechanism of gibberellin metabolism during fruit development and provide scientific theory for the production of seedless loquat via hormone and molecular level.The main results are as follows:1.Determination of ten kinds of plant hormones during Loquat fruit development?1?We established a high-performance liquid chromatography method for determination of ten phytohormones?trans-zeatin,gibberellin A3,kinetin,salicylic acid,6-benzylaminopurine,benzoic acid,indole-3-acetic acid,abscisic acid,indole-3-butyric acid and jasmonic acid?simultaneously in loquat?Eriobotrya japonica?Thunb?Lindl.?flowers and fruitlet.Ten phytohormones were detected by the following conditions:reversed-phase column Dikma C18?250 mm×46 mm,5?m?with the temperature at 35?,methanol /acetonitrile /phosphate buffer?pH= 3.0??20:20:60,vol/vol/vol?mixture was used as mobile phase with flow rate of1 m L·min-1,210 and 254 nanometer were elected as determination wavelength.The standard calibration curves of ten phytohormones all show high linearity,and the correlation coefficients are all above 0.9915.Limits of detection of all ten phytohormones range from 0.004 ng to 4.92 ng,with relatively higher precision?%RSD?=?0.026%–3.05%?and accuracy?%recovery?=?58.6% and 85.2%?,suggesting that this method is an efficient tool for routine screening of phytohormones contents in plants.We used this method to monitor the changes of phytohormones contents during the development of loquat fruitlet,Lycopersicon esculentum?tomato?and Brassica oleracea L.tender leaves also be used to verify the efficiency of the this method.?2?The content of GA3 and SA in the process of loquat fruit development was higher than that of other plant hormones.Futher more,there were two obvious ABA peaks in diploid and triploid,which were similar to those of fruit dropping time.2.Key enzymes in gibberellin pathway of loquat were cloned?1?The GA20ox1 gene fragment has a 1083 bp length,nucleic acid sequence analysis showed that : the highest similarity is 99% with apple GA20ox1?XM₀08344743?;GA20ox2 gene fragment has a 1122 bp length,nucleic acid sequence analysis showed that : the highest similarity is 98% with apple GA20ox2?LOC103439689?;GA3ox gene fragment has a 931 bp length,nucleic acid sequence analysis showed that the highest similarity is 98% with pear GA3ox?LC013247.1?,followed by apple GA3ox1?LOC103429342?is 98%;GA2ox gene fragment has a904 bp length,nucleic acid sequence alignment and analysis the highest similarity is98% with pear GA2ox1?LOC103957332?;CYP714A gene fragment has a 797 bp length,nucleic acid sequence analysis the highest similarity is 99%with apple CYP714A1?LOC103438620?;CYP714C gene fragment has a 692 bp length,nucleic acid sequence analysis the highest similarity is 97% with apple CYP714C2?LOC103417797?,followed by pear CYP714C2?LOC103946398?is 96%.?2?The cDNA sequences obtained by homologous gene cloning were analyzed by local blast with the nucleic acid database of the loquat transcripts.Three full-length cDNAs and three cDNA fragments of the target genes were obtained.The three full-length sequences were GA20ox1,GA2 ox and CYP714 C.The cDNAs of the fragments sequences were GA20ox2,GA3 ox and CYP714 A,respectively.3.Gene expression of the key enzymes in loquat gibberellin metabolism pathway were analyzisedAccording to the results from Dawuxing and A322 hormone determination on the day of anthesis?0d?and 100 mg/L GA3,water?0.1% Twain-80?and 1d,3d,5d,15 d,25d and 30 d after treatment in each period.And we use the qRT-PCR technology to express characteristics of the key enzyme genes of gibberellin metabolism pathway.The results showed that the expression of GA20ox1 and GA3 ox gene was significantly changed by gibberellin treatment.However,the expression of other genes did not change very much.The expression level of GA20ox1 gene was the highest at 30 d after treatment with GA3,and the lowest was at 5d after treatment.The expression level of GA20ox1 gene was the highest at flowering day in A322,and the expression change from 1d to 30 d was not significant.The expression of GA3 ox gene in Dawuxing was higher than that in A322.In the Dawuxing,the expression of GA3 ox gene reached the maximum at 1d after treatment with GA3,then the expression decreased;In A322,the expression of GA3 ox gene reached the maximum at 1d after treatment with GA3,and maintain stable from 5d to 30 d,with the expression decreasing slowly.