Functional Studies On The LaMIR166a Overexpression During Somatic Embryogenesis Of ESMs And Germination Processes In Larch

The somatic embryogenesis(SE)system is an ideal model system for molecular genetic improvement of conifers,to study the early developmental regulation and morphogenesis of gymnosperms.However,Larix spp.SE system is still limited in the production practice due to the problems occurred such as the high rate of deformity and low germination rates.In the previous study in our lab,embryogenic cell lines of the endogenous LaMIR166 a overexpression and the auxin polar transport inhibitor N-1-naphthylphthalyl(NPA)exogenous treatment revealed similar phenotypes that abnormal development of mature somatic embryos,suggesting that there is a regulatory relationship between miR166 and its target genes LaHDZ31-34 and auxin signaling pathway in SE of Larix leptolepis(L.leptolepis).In the present study,culture from pro-embryo to germinated seedlings of S287 embryogenic cell line were collected as the material,and the function of LaMIR166 a overexpression during the SE of Embryogenic Suspensor Mass(ESMs)and germination processes in larch were studied,and the regulation of miR166 a on IAA signaling pathway was also discussed.The following results were obtained:(1)Agrobacterium-mediated genetic transformation method was used to transform LaMIR166 a overexpression vector into Embryogenic Suspensor Masses(ESMs)of L.leptolepis and 6 resistant cell lines were screened and verified by qRT-PCR detection.The further data statistics and observation demonstrated that overexpression of LaMIR166 a markedly lead to slower proliferating rates,higher deformities of somatic embryos,higher germination rates and denser lateral roots of regenerated seedlings.(2)The key enzyme genes of indole-3-acetic acid(IAA)signaling pathway LaNIT and LaARF were identified,and their expression pattern were analyzed.qRT-PCR analysis showed that expression levels of LaNIT,LaARF1 and LaARF2 were upregulated at early stages during somatic embryo development.IAA contents were detected by liquid chromatograph-mass spectrometer(LC-MS),and the results showed that IAA levels increased at early stages of somatic embryo development,suggesting that LaNIT was involved in IAA biosynthesis and IAA signaling pathway play vital roles during somatic embryo development of L.leptolepis.(3)qRT-PCR results revealed that the expression pattern of LaNIT was similar to that of LaHDZ31-34,which showed sharply up-regulated expression at early stages during embryogenic development,indicating the positive regulatory role of LaHDZ31-34 on LaNIT.Expression of HD-ZIP Ⅲ were not wholy downregulated by miR166 a but a dynamic pattern occurred during SE process in larch,suggesting that regulation of HD-ZIP Ⅲ by miR166 a exits stage specific characteristics.(4)A network model of miR166a/ HDZIP Ⅲ regulation of somatic embryonic germination of Larch was proposed.It has been found that miR166 regulate HDZIP Ⅲ at posttranscriptome level,and HDZIP Ⅲ has a positive regulatory effect on La NIT.The changes of LaNIT expression leads to IAA levels variation,which further affect LaARF1 and LaARF2 expression levels and ultimately influences the somatic embryos germination of L.leptolepis.(5)Agrobacterium-mediated genetic transformation method was used to transform LaMIR166 a overexpression vector into tobacco leaves,and 3 transgenic lines were obtained after qRT-PCR detection.Growth parameters like the root length and base diameter,lateral root density,stem ground diameter and stem internode length in LaMIR166 a overexpression lines were significantly higher than those of WT plants.Microscopic observation of stem slice and root squash showed that overexpression of LaMIR166 a significantly promoted xylem formation and development and root hair growth,which also along with bend phenotype.The regulatory role of LaMIR166 a on tobacco plants might be correlated with IAA signaling pathway.(6)Two CRISPR/Cas9 vectors were constructed specific for LaMIR166 a and LaHDZ31.Agrobacterium tumefaciens-mediated genetic transformation was carried out and 33 resistant cell lines were obtained.PCR detection of resistence marker gene HPT proved that the vectors were all transformed into embryogenic cell lines of L.leptolepis.Further enzyme cutting of cell lines with pZJP15 revealed that transgenic lines appeared to have positive results.Sequencing results of PCR products were remain to be seen.In conclusion,miR166/HD-ZIP Ⅲ play vital regulatory roles in somtic embryogenesis of L.leptolepis.These results might provide scientific basis for further study on the molecular regulation mechanism of somatic embryogenesis in coniferous trees and obtain efficient and stable stem cell breeding system in L.leptolepis.