Phytohormone Content Detection And Expression Analysis Of Aux/IAA Gene After Gibberellin Treatment Of Triploid Loquat

The triploid loquat is difficult to form normal pollen due to abnormal gametophyte meiosis,resulting in pollination and fertilization failure.The triploid loquat has a low fruit setting ratio,but fruit setting ratio is significantly increased after the application of exogenous gibberellin.Studies have shown that auxin is one of the main plant endogenous hormones that promote the fruit set and fruit growth and development.The main function of auxin is to promote cell division.The Aux/IAAs family gene is a key factor in the feedback regulation of auxin.In order to discover the change of the auxin signal transduction pathway during exogenous gibberellin-induced fruit set in physiological and molecular,this study determined the changes of endogenous auxin content,auxin response gene expression and thickness of ovary wall after exogenous gibberellin treatment,aimed to study of the mechanism of triploid loquat furit set.In this study,triploid loquat strain of A322 used as the plant materials.The control uses water treatment,analysis the changes of fruit set,thickness of ovary wall,the content of six endogenous hormones?IAA,ABA,GA₃,ZT,KT and SA?in triploid loquat ovary and other parts after exogenous gibberellin treatment.Based on previous transcriptome,we cloned six Aux/IAA family gene:IAA6,IAA8,IAA9,IAA10,IAA11and IAA20,and further analyzed the expression of these six genes in the triploid loquat fruit set.Main results are shown as following.1.After exogenous GA₃ treatment,the triploid loquat fruit setting rate was significantly increased,and the average fruit setting rate reached 43.32%,while the fruit setting rate in the control group was only 1.9%.Morphological observation of the flower showed that the sepals began to close after 7 days after GA₃ treatment,and totally closed after 21 days after treatment,indicating successful fruit set.Paraffin sections showed no significant changes in the ovary and surrounding tissue cells during the fruit setting period.The ovary wall thickness was not significantly thickened,indicating that the cell division and expansion were slow at this time,belonging to slow growth period.Comparied the loquat ovule longitudinal and transverse diameter during fruit set,the ovules were found no development in both treatment group and control group.2.The detection of hormones of the triploid loquat during the fruit setting period were found that the content of IAA in the ovary was significantly higher than that in other parts after exogenous GA₃ treatment,and there were two high peaks.The increase of auxin may be related to exogenous GA₃ treatment,and the high concentration of IAA replaced fertilization to initiate triploid loquat fruit set.After exogenous GA₃ treatment,ABA content was low in early period,while the water treatment group showed a peak at 21 days,which was consistent with the flower drop.In addition,the content of ZT in ovary and other parts,was higher than the other five hormones,so we speculated that the endogenous ZT may be related to the loquat fruit set.3.Nucleic acid sequence analysis showed that:the similarity between the IAA6and MdIAA6?HM122447.1?was 93%;the similarity between IAA8 and MdIAA8?NM₀01294015.1?was 97%;the similarity between IAA9 and MdIAA9?NM₀01293892.1?was 93%;the similarity between IAA10 and MdIAA10?HM122440.1?was 90%;the similarity between IAA11 and MdIAA11?NM₀01293857.1?was 97%;the similarity between IAA20 and MdIAA20?NM₀01293857?was 95%.The result indecated that the six genes were what we wanted.4.Real-time PCR analysis showed that the expression of the six genes changed after GA₃ application.The expression level of IAA6,IAA8 and IAA10 in ovary were significantly higher than that in other parts,IAA9 expression level was similar in ovary and other parts,while IAA11 and IAA20 expression level were less in the ovary than in other parts.In addition,at the same developmental stage,the expression of IAA9 in the ovary was consistent with the change in endogenous IAA content after exogenous GA₃treatment.The endogenous auxin content increase,induced the expression of IAA9up-regulation,thereby induced the loquat fruit set.In the ovary,IAA8 had been highly expressed at 3d after exogenous GA₃ treatment,suggesting that function of IAA8 may be similar to IAA9.