Dormancy Characteristics And WRKY Gene Analysis Of T.thianschanica Seeds

Tulipa thianschanica is a bulbous flower native to China and an important germplasm resource for tulip cross breeding.However,tulip seeds are mostly dormant,which limits their utilization.In this study,the dormancy of tulip Tianshan seeds was broken at 4℃ under low temperature and humidity and on the basis of applying exogenous plant hormones ABA and FLU,and the dormancy breaking characteristics of seeds were observed.UPLC-MS/MS was used to measure the content changes of ABA and GA₃in seeds during dormancy breaking with different treatments,and how some genes in WRKY gene family responded to ABA signals was also studied.Firstly,bioinformatics analysis,phylogenetic tree construction analysis and electrophoresis detection were carried out on the screened gene family members.Finally,four WRKY transcription factors related to seed dormancy of Tulip in Tianshan Mountains were screened out,and the expression patterns were analyzed to analyze the expression characteristics of these four genes in different germination processes.This experiment explored the hormone regulation mechanism of breaking dormancy of tulip seeds in Tianshan Mountains,and laid a theoretical foundation for further accelerating tulip breeding process.The results:（1）4℃wet condition is the best condition to break the dormancy of tulip seeds in Tianshan Mountains in advance.The water content and water absorption of seeds reached the peak in about 8 hours.The longer the seeds are stored,the lower the germination rate.After treated with 100μmol/L ABA,the growth rate of embryo length slowed down,and the germination time was delayed and the germination rate was reduced.However,after 50μmol/l LU treatment,the growth rate of embryo length is accelerated,which will advance the germination time and improve the germination rate.（2）UPLC-MS/MS was used to determine ABA and GA₃ in tulip seeds of Tianshan Mountains.The content of ABA and GA₃ was the highest when the sample was extracted with 80% methanol.Although the use of extraction column can purify impurities with certain effect,it will significantly affect the measured content,so the extraction column is not used.The system of 0.1% formic acid water and methanol was selected as chromatographic conditions,and the chromatographic peak type and total ion flow diagram of mass spectrometry were satisfactory.Mass spectrometry conditions ABA and GA₃ are suitable for negative ion mode.In the process of breaking dormancy of tulip seeds in Tianshan Mountains,ABA content increased first and then decreased,and GA₃ decreased first and then increased at 4℃.After stratification at 4℃ and 100μmol/L ABA treatment,ABA content in seeds increased greatly,and GA₃content reached the highest value after 20 days.After stratification at 4℃and 50μmol/l LU treatment,ABA content in seeds decreased significantly,while GA₃ decreased first and then increased.（3）Through transcriptome data,qRT-PCR（real-time fluorescence quantitative experiment）was performed on four WRKY genes.The results showed that the expression levels of TgWRKY4,TgWRKY24 and TgWRKY28 all reached the highest value on the 30th day,and were relatively low on the 50th day.The expression was related to the changes of ABA content in plants,and there was a trend of increasing at first and then decreasing.It is concluded that these three genes are positive regulators of ABA signal response.However,the expression of TgWRKY23 decreased gradually.