Molecular Cloning And Promoter Function Analysis Of Gene MIR171 Relevant To Lily Somatic Embryogenesis

As a regulatory factor of gene expression,miRNA plays an important role in plant somatic embryogenesis.In our previous studies,lpu-miR171a and lpu-miR171b were identified for the differently expression between embryogenic callus and non-embryogenic callus of Lilium pumilum DC Fisch.native to China.Based on the previous work,we continued to research the regulation role of miR171 in Lilium regeneration in vitro,clone primary transcription of MIR171 gene and its promoter sequence.And then,we analyzed the temporal-spatial expression patterns of miR171 genes by promoter-GUS reporting system.The mechanism of the miR171 regulating somatic embryogenesis in transcription level was discussed.The main test results were as follows:（1）Using sterile scales as explant,somatic embryogenesis were successfully induced by direct regeneration and indirect regeneration for Lilium pumilum DC Fisch..MS+4.0μmol·L^-1 PIC+0.01μmol·L^-1 ABA can induce somatic embryogenesis directly.MS+4.0μmol·L^-1 PIC+25.0μmol·L^-1 SA may induce embryogenic callus formation.Both induction rates can reach 100%.Different approaches of somatic embryogenesis vary greatly.For direct somatic embryogenesis,the spherical embryos may induce from scales directly,then go through a long-term heart-shaped embryo phase and a short-term torpedo-shaped embryos phase,and ultimately giving rise to cotyledon-shape embryos.For indirect somatic embryogenesis,after the embryonic callus differentiated into the spherical embryo,embryogenic cultures go through a long-term torpedo-shaped embryo phase to enter the cotyledon-shape embryos period,but the heart-shaped embryo is almost invisible.The expression of miR171 is different in the two pathways of regeneration.In the embryogenic acquisition stage of somatic embryogenesis,as the induction rate of somatic embryos increased,the expression of miR171a/b decreased,while the expression of target gene SCL6increased significantly.In the adventitious bud primordium formation stage of organgenesis pathway,the expression of miR171 was significantly increased.From above analysis,miR171involved in the process of embryogenic acquisition by regulating the targets SCL6,during somatic embryogenesis.（2）Primary transcription is the initial product of MIR gene transcription,contains a short open reading frame,and can improve its specific miRNA expression in transcription levels by encoding short peptides.lpu-pri-mir171a and lpu-pri-mir171b were cloning with RACE technology,and the length were length 593bp and 404bp,separately.The primary transcription of the miR171 family was less conservative.The distances between pre-mir171a and pre-mir171b to transcription initiation sites（TSS）were 69 bp and 22 bp,to 3’polyA were326 bp and 239 bp,separately.In the sequence from TSS to pre-miR171,there contain 3 and1 short ORF,separately.（3）Using FPNI-PCR technique,1450 bp and 953 bp promoter fragment in the upstream of lpu-pri-miR171a and lpu-pri-miR171b were cloned.In both sequences,there are core promoter elements such as TATA-box,CAAT-box,and a number of identical or similar transcription factor binding sites.It may conclude that the 5’upstream flank sequence of MIR171a and MIR171b has the ability to promote the downstream gene expression.These two miRNAs have independent transcription units,and the regulation mode between different members of gene family was similar in transcription.A large number of cis-acting elements are concentrated in the upstream-1 kb of TSS,mainly consist of mesophyll tissue,root,anther and seed-specific expression elements,and the element responded to light,abiotic stress,and various phytohormone such as cytokinin,abscisic acid,jasmine acid,salicylate,ethylene,etc.It is implied that,the regulation of MIR171 is complex and may be affected by multiple signaling pathways.In addition,the MIR171 gene promoter sequence contains a large number of glucose and starch metabolic response elements,indicating that MIR171 take part in carbohydrate metabolism.（4）According to the prediction results of full length sequence and cis-acting element of MIR171a promoter,five deletion promoter fragments with different lengths was cloned.The full length and deletion promoter fragments of the MIR171a gene were connected with the GUS gene to construct the plant expression vector,and then transformed into Arabidopsis thaliana successfully by dipping flower method.After resistance selected,the transgenic Arabidopsis thaliana was obtained.The histochemical analysis of GUS protein in T3transgenic plants showed that,the sequence in the upstream 5’region of the MIR171a gene have promoter activity,which could drive the expression of GUS gene in cotyledons and young roots of Arabidopsis thaliana seedling.The expression of GUS can also be detected in the initial euphylla under the stress of highlight.The core area of MIR171a gene promoter was in 232 bp of the upstream of the transcription sites.There may be specific enhancer element in the range of-650 bp to-823 bp of TSS.Driven by MIR171a promoter,GUS gene was expression in different tissue and time.The content of GUS was the highest in seed germination,and then gradually declines as the growth.15 days after germination,GUS was completely undetectable.According to the function of MIR171a promoter and the expression pattern of miR171a in lily somatic embryogenesis,it is concluded that miR171a may participate in the occurrence and differentiation of the meristem,especially cotyledon primordium.（5）MIR171a gene promoter can respond to light,abiotic stress and a variety of hormone signals.The content of GUS increased under the condition of strong light stress and illumination variation.The response region of light is located in-823 bp to-1059 bp area of TSS.The expression of GUS was significantly increased in the condition of salt and drought stress,,and slight increase in low temperature condition.Multiple hormones can affect MIR171 promoter activity.The expression of GUS was expressed by BA,and increases with stress response hormone,such as MeJA,ABA and SA.In view of plant somatic embryogenesis always accompanied with stress responses,we conclude that miR171a regulate the somatic embryogenesis of Lilium by participating in stress response.