Molecular Cloning And Functional Analysis Of MiR164b And Its Target Gene PeNAC1 From Phyllostachys Edulis

Stress is one of the main factors affecting the growth, development and formation of biological production and quality of plants. To adapt to the environment, a set of self-regulatory mechanisms was formed in plants, among which miRNAs regulated their target genes mainly through degradation at transcript level or inhibition at translation level.. MiR164 b was specific for plants. Main targets of miR164 b were genes encoding NAC transcription factors, which played important roles in regulation of plant growth and development, as well as response to abiotic stresses such as salt and drought. Therefore, it is important to focus on functional studies of miR164 and NAC transcription factors for revealing their regulation manners and roles in the process of resilience. Phyllostachys edulis was selected for isolation of sequences related with ped-mi R164 b and its target PeNAC1. Based on the analysis of gene structure characteristics, the validation of the target site in PeNAC1 by ped-mi R164 b was performed with RLM-RACE. The expression patterns of ped-miR164 b and PeNAC1 were analyzed using real time quantitative PCR. Meanwhile, the functions ped-mi R164 b and PeNAC1 were also verified through transgenic technology. The results provide a reference for bamboo molecular breeding for resilience. Main results are as follows:1. Gene Isolation and bioinformatic analysisThere are two members(ped-miR164 a and ped-miR164b) of miR164 in Phyllostachys edulis(BambooGDB, www.bamboogdb.org). We got the precursor sequence of ped-miR164 b through PCR with specific primers designed according to PH01000543, which was 82 bp and could form a stable stem-loop structure in the predicted second structure with mature sequence(5′-UGGAGAAGCAGGGCACGUGCU-3′) on the 5’ end of the arm. Meanwhile, there was only last base in the mature sequence of ped-miR164 b different from the standard sequence of miR164 family(Aâ†'U), indicating that the mature sequences of miR164 were highly conserved.Based on the BLASTn result of ZmNAC1 sequence, a homologous gene(FP095491) was abstained from the full length cDNA database of Ph. edulis. Sequence analysis showed that there was a target site of miR164b(5′-AGCAAGTGCCCTGCTTCTCCA-3′) at the 3′ end of ORF in FP095491. Specific amplification was carried out for the ORF which was 867 bp encoding a protein with 288 amino acids. The protein had high similarity(more than 94.52%) with NAC1 proteins of other plants such as Oryza sative, Zea mays and Arabidopsis thaliana, which had five specific domains(A, B, C, D and E) of NAC1. These results indicated that FP095491 was a gene belonged to NAC family and named as PeNAC1.2. Validation of the cleavage positionThe target site of ped-miR164 b in PeNAC1 was validated through RLM-5′ RACE. The sequencing result demonstrated that there were nine of ten samples with the cleavage at the 10 th and 11 th bases, indicating that ped-miR164 b could regulate its target gene PeNAC1 at transcript level. This result was consistent with that in Zea mays, which further verified that the target site of miR164 family was conserved.3. The temporal and spatial expression of genesThe result of tissue specific expression showed that ped-mi R164 b and PeNAC1 were constitutively expressed, with the highest level of ped-miR164 b and the lowest level of PeNAC1 in root. Real-time quantitative PCR analysis showed that ped-miR164 b was down-regulated under the treatments of NaCl(300 mmol·L-1), low temperature(4℃) and high light(1500 μmol·m-2·s-1), but it was up-regulated obviously under the treatment of GA3(100 μmol·L-1). Therefore, the results indicated that ped-miR164 b and PeNAC1 played important role in response to the stress- and hormone-induced process.4. Ectopic expression of genes and phenotype analysis of transgenic plantsThe sense and antisense vectors of PeNAC1 as well as an over-expression vector of ped-mi R164 b precursor were constructed and transferred into model plant Arabidopsis thaliana respectively. The results indicated all the genes were expressed in transgenic plants confirmed by RT-PCR. The phenotype analysis demonstrated that some cotyledons and petioles were fused to different degree, among which some cotyledons developed into cup-shaped or dissymmetric, notched blades in the ped-mi R164 b transgenic lines. In addition, the number of root was reduced compared with that of wild type. The sense transgenic lines grew fast and had more lateral roots than wild type. The antisense transgenic lines grew slow and short, among which some lines had similar phenotype to those of ped-miR164 b precursor transgenic lines with less notched or dissymmetric blades, short roots and less lateral roots.5. Resistance analysis of transgenic linesThe transgenic plants were treated with NaCl(300 mmol·L-1) and PEG6000(30%) respectively. The survival rate was recorded and the chlorophyll fluorescence parameters of transgenic plants were measured with the wild type as controls. The results showed that the survival rate of sense transgenic plants was 80-90%, and the value of Fv/Fm was reduced slowly. On the contrary, the survival rate of ped-miR164 b and antisense transgenic plants was only 20-30%, and the values of Fv/Fm were decreased rapidly. Based on this collectively results, it was inferred that interaction between ped-miR164 b and its target gene PeNAC1 might influence the ability of transgenic plants resistant to salt and drought stresses through regulating the development of roots.6. Functional study on regulatory sequence upstream mi R164 b precursorThe upstream sequence of ped-mi R164 b precursor was obtained by PCR and analyzed with online software(Plant CARE). The results showed that the sequence was 1,480 bp containing many of response elements responsive to stresses, such as ARE, LTR, MBS and so on, besides the basic regulatory elements such as TATA-box and CAAT-box. The result implied that ped-mi R164 b might participate in the regulating processes of stresses.The expression vector of upstream sequence of ped-mi R164 b precursor fused with GUS was constructed and transferred in A. thaliana, which were selected with Kan(50 mg·L-1) and strained with GUS reagent. The result demonstrated that root, stem and leaf were stained with blue, which indicated that this sequence could function as a promoter and express constitutively.MiR164 family and NAC transcription factor family were specific for plants. The results showed that ped-miR164 b and PeNAC1 were constitutively expressed, and participated in the regulatory processes of hormones and abiotic stresses, in which ped-miR164 b regulated PeNAC1 through cleavage at the 10 th and 11 th bases of the targeted site. Overexpression of ped-mi R164 b and PeNAC1 in A. thaliana affected the morphogenesis of leaf and root. The physiological index of transgenic plants indicated that resulting in different abilities of resistant to drought and salt which indicated that ped-miR164 b and PeNAC1 might play important roles in the regulatory process under drought and salt. These results provided gene resources for the molecular breeding of bamboo resilience.