Evolutionary Characteristics And Expression Patterns Of MiR172 Members In Chinese Kale

Chinese kale is an annual vegetable belonging to Brassicaceae.It originated in the southern part of China.The microspores are an important part of sexual reproduction and play important roles in making double haploids.Studies in model plants have shown that siRNA participate in plant development by regulating its target genes,and the role of miR172 in floral bud development has been elucidated.In this paper,the correlation between microspore development and floral bud parameters was explored in different genotypes of Chinese kale.The evolutionary characteristics of miR172 family members were analyzed,and the expression of pre-miR 172 family members were analyzed by qRT-PCR.According to the expression of different members in various floral organs,pre-miR172a-5p with high expression in anthers was screened as the next direction.The expression of pre-miR172a-5p in different developmental stages of microspores was further analyzed.The 5’ UTR and 3’ UTR of the Chinese kale pri-miR172a-5p were amplified by RLM-RACE technology.The main findings include the following four aspects:1 Link the developmental stage of microspores to floral organ parameters of Chinese kaleThe kale resources were collected,and main cultivar were planted in Fujian Province and the time required for vegetative and reproductive growth was observed.The cytological observation of microspores’development process was obtained by DAPI fluorescence staining.Parameters of flower bud including its longitudinal diameter,transverse diameter,aspect ratio,length of the stalks and so on were investigated.The results showed these parameters were different in microspore developmental stages,and the relation of microspores’ developmental stages and growth parameters was established by the length of petals,longitudinal diameter of anther,transverse diameter of anther and the ratio of petals to anthers.The diameter of sepal,transverse diameter of bud and longitudinal diameter of anthers are significantly different among varieties,which is suitable as a reliable indicator for judging the development of microspores.By combining these indicators with the other external morphological features of flower buds and anthers,it is possible to infer the development stage of microspores quickly,accurately and conveniently from the external morphological features of flower organs.2 Analysis of evolutionary characteristics of miR172 members and their expression patterns in Chinese kaleIn order to understand the evolutionary characteristics of miR172 family members and their expression patterns in different tissues were analyzed.The results showed that:(1)There were 20 miR172 members in Chinese kale.By comparison,all 20 sequences have an overlapping region,in which 8 bases of ACTAGATC are highly conserved.(2)The results of mfold prediction showed that folding free energy of five precursor components of miR172a family ranges from-54.55 kal/mol to-78.60 kal/mol,both of which could spontaneously form a typical and stable stem-loop secondary structure.(3)Phylogenetic tree shows that the MIR172 in Chinese kale is closely related to papaya MIR172.(4)A total of 13 different genes were predicted to be targets of miR172a in Chinse kale and multiple members could also target the same gene..(5)The expression of different members of pre-miR172a and pre-miR172b was significantly different in different tissues.The pre-miR172a-1,pre-miR172a-2,pre-miR172a-3 and pre-miR172b was highly expressed in pods of Chinese kale and the pre-miR172b-5p and pre-miR172b-5p2 was highly expressed in flowers,indicating that miR172 plays an important role in the development of Chinese kale flowers and pods.3 Differential expression analysis of 10 pre-miR172 in floral organs of Chinese kaleThe qRT-PCR primers were designed based on the pre-miR172 sequences,followed by RNA extraction from the floral organs of ’Fuzhou Huanghua’ and ’Lvbao’.The gene expression level was examined.The results showed that the expression of pre-miR172a and pre-miR172b family members in pistil,stamen,petal and sepal were all specific,and some genes expressed differently in two varieties.The expression of 10 members in flower organs of ’Fuzhou Huanghua’ was the highest in anthers,followed by sepals and pistils.In anthers,the relative expression of pre-miR172a-5p was the highest,followed by pre-miR172b-5p4;In sepals,the expression of pre-miR172a-4 was the highest,followed by pre-miR172a-1;In pistils,the expression of pre-miR172a-5p was the highest,followed by pre-miR172b-5p3.The expression of pre-miR172 members in ’Lvbao’ was the highest in pistils,followed by anthers and flower.The relative expression of pre-miR172b-5p3 was the highest in pistil,followed by pre-miR172b-5p;the expression of pre-miR172a-5p was the highest in anther,followed by pre-miR172b-5p3;the relative expression of pre-miR172b-5p3 was the highest in flower bud,followed by pre-miR172a-3.The expression levels of pre-miR 172 members were the least in both petals and filaments.Therefore,members of the pre-miR 172a and pre-miR 172b family play a role in the formation and development of the anther and pistil of Chinese kale,and each family member has a different expression pattern.4 Expression patterns of pre-miR172a-5p in microspores’different developmental stages of Chinese kaleIn order to further study the function of pre-miR172a-5p in the development of microspores,its expression in different microspore developmental stages of Tuzhou Huanghua’,’Cuibao’ and ’Jinpin 1290’was analyzed.In the three varieties of Chinese kale,the expression of pre-miR172a-5p reached the highest in the early and middle stages of mononuclear.With the further development of microspores,the expression of this precursor showed an overall downward trend.The expression levels of pre-miR172a-5p were different in different varieties,and the expression level of pre-miR172a-5p was the highest in the early and middle stages of‘Fuzhou Huanghua’,followed by‘Cuibao’and‘Jinpin 1290’.From kale transcriptome database access to the pre-miR172a-5p sequence,design 5’ and 3’ RACE inside and outside primers,by RLMRACE technique,two 5’ UTR of candidate primary were obtained and two transcription initiation sites were found for pri-miR172a-5p.Further analysis of the cis-acting elements of the promoter before the transcription start site(2000bp)was conducted,and it was found that the promoter had the functional elements related to the expression of light,hormone,abiotic stress,anaerobic and meristem,providing a theoretical basis for further study of miR172’s function in Chinese kale.