Study On Fine Expression Characteristics Of Key Genes Of Pistil Development In Japanese Apricot(Prunus Mume Sieb.et Zucc.)

Prunus mume(Prunus mume Sieb.et Zucc.)is a perennial drupe of the genus Prunus in the family Rosaceae.Pistil is the quality and basis of fruit formation.The phenomenon of pistil abortion in Japanese apricot restricted seriously the yield of fruit and the development of Japanese apricot production.Our previous study showed that pistil abortion was related to PmTCP4,PmARF1 and auxin IAA.In order to reveal the molecular mechanism of pistil abortion,we established a technical system of paraffin section combined with laser to capture and extract micro RNA firstly based on previous studies.Then,the flower buds of Japanese Apricot varieties‘Longyan’and‘Daqiandi’were used as materials to make paraffin sections.The stigma,style and ovary of the pistil of the two varieties were respectively cut with the constructed system,and the trace RNA was extracted to verify the expression of Pm TCP4 in different parts of the two cultivars.Finally,we picked out Auxin Response Factor PmARFl according to the transcriptome sequencing results of flower buds of the two varieties,and then the expression pattern of PmARF1 gene in the flower buds of different varieties of‘Longyan’and‘Daqiandi’at different periods was detected by qRT-PCR.We analyzed the regulatory relationship of the gene in the development of Japanese Apricot pistil by comparing the expression levels of the gene in two materials.The main results of this study are as follows:1.In order to solve the problems that the pistil of the Japanese apricots too small,it is difficult to separate the tissues,and the trace RNA is not easy to be extracted,we have established a technology system suitable for the use of laser capture microdissection to extract trace RNA.Using the flower buds and pistils of Japanese Apricot varieties‘Longyan’and‘Daqiandi’as experimental materials to make paraffin sections,and then compare the cutting effects of the strips of different thickness.Select the optimal strip thickness to make sections.The Leica LMD 7 000 laser capture microdissection system was used as the core of the experiment.We compare the cutting effects of different laser intensities select and the laser intensity with the best cutting effect.The pistils of two Japanese apricot varieties were cut separately.We compared the quality of RNA extracted from the samples after normal temperature storage and liquid nitrogen storage.Finally,an optimal system for extracting trace RNA with laser capture microdissection of Japanese Apricot was established.During the paraffin section making process,the part we improved was that the test materials should be stored in a 4℃ refrigerator at low temperature.Other steps such as dehydration and transparency were performed in a 4℃ refrigerator,and then make slice.We have successfully determined that the optimal strip thickness of paraffin sections was 10μm,and the best laser intensity for laser capture microdissection was 30,and It was also clear that the RIN value of RNA extracted from the cutting material stored in liquid nitrogen after cutting was higher than that stored at room temperature.2.In order to study the differential expression of stigma,style and ovary during the development of pistil in PmTCP4,we used paraffin section combined with laser capture microdissection to separate the stigma and style and ovary of‘Longyan’and‘Daqiandi’pistil.Then,we used the cutting material to extract trace RNA and analyzed the role of PmTCP4 in the development of Japanese apricot pistil by qRT-PCR.We cut different parts of the pistils of the variety‘Longyan’and‘Daqiandi’by the previous methods and compared the expression of PmTCP4 in different parts of the pistil of the two varieties.The expression level of PmTCP4 in the pistil of‘Daqiandi’was higher than that in‘Longyan’,and the expression level was the highest in the middle of December,especially in the style of the pistil of‘Daqiandi’,it indicated that the high expression of PmTCP4 stimulated the abortion of the pistil of Japanese apricot,and the transcription factor of PmTCP4 was a negative regulatory gene.In this study,the primary role of PmTCP4 in the development of the pistil of Japanese apricot was explored,which provided a theoretical basis for the subsequent molecular biology study of the pistil of prune and other woody plants.3.ARF transcription factors in plants are important regulators of the auxin signal transduction pathway.Auxin response factor,as an important auxin regulator,may play a very important role in the development of Japanese Apricot pistil.The pistil abortion of‘Daqiandi’was very serious,the proportion of imperfect flowers was up to 76%,while the proportion of‘Longyan’imperfect flowers was only 5%,the pistil development was relatively complete.To investigate PmARF1 of flower buds development effect,we picked out auxin response factor PmARF1 based on the transcriptome sequencing results of the two varieties,and then the expression pattern of PmARF1 gene in the flower buds of different varieties of‘Longyan’and‘Daqiandi’at different periods was detected by qRT-PCR.By comparing the expression of the gene in the two materials,the regulatory relationship of the gene in the development of pistils was analyzed.According to the results of the study,it was found that PmARF1 gene was expressed in the flower buds of’Longyan’ and ’Daqiandi’ from November 17 to March 1 of the following year,the expression of PmARF1 gene was significantly up-regulated in the flower buds of ’Longyan’and ’Daqiandi’ in November and December,while the expression in the flower buds was significantly down-regulated and gentle in January,the expression of this gene in the flower buds of ’Daqjiandi’ was significantly higher than that of ’Longyan’ flower buds in November and December,It showed that November and December may be important periods for pistil development in Nanjing.At the same time,it also showed that PmARF1 gene played an important regulatory role in the all period of flower development of’Daqiandi’.During the growth of the variety of ’Longyan’,PmARF1 showed a negative correlation with auxin IAA;during the growth of the variety of ’Daqiandi’,PmARF1 and auxin showed a basically positive correlation,it indicated that PmARF1 gene inhibit the pistil normal development of ’Daqiandi’,which can aggravate the phenomenon of abortion of Japanese apricot pistil.It may be a negative regulatory gene for the normal development of Japanese apricot pistil.