| Cold stress seriously restricts the distribution and yield of citrus industry.Genetic engineering is an effective mean to cultivate cold-resistant varieties of citrus.While mining and identifying the mechanism of cold tolerance-related genes is the precondition and the key of breeding.C3H-type zinc finger protein is one of the largest families of plant transcription factors,which participates in various biological processes,such as seed germination,leaf senescence,flower development and abiotic stress response.Currently,the whole genome of C3 H zinc finger protein family has been analyzed in many plants,but the function and regulation mechanism of most of them are still unclear.In the early stage of this experiment,a tandem C3 H zinc finger protein PtrTZF1,which can respond to low temperature rapidly and continuously,was screened and cloned from trifoliate orange,and its expression profiles under low temperature was analyzed.We got the transgenic tobacco overexpressing of PtrTZF1.Then we preliminarily identified the cold-resistant function of PtrTZF1 and its regulation mechanism under low temperature.The research results are as follows:1.A tandem C3 H zinc finger protein without intron was cloned from trifoliate orange.The ORF length was 1968 bp,encoding 655 amino acids,isoelectric point was 6.5,and the molecular weight of the protein was 71.84 k Da.The expression profiles of PtrTZF1 under low temperature was analyzed by real-time fluorescence quantitative PCR.It was found that the expression of PtrTZF1 was the highest at 24 h,reaching about 48.4 times compared with 0 h;After 6 hours of low-temperature treatment in lemon,the expression level slightly increased,barely up to 6.4 times.The subcellular localization of PtrTZF1 was analyzed by transient expression in tobacco,and found that PtrTZF1 was localized in the nucleus and cell membrane.2.PtrTZF1 was overexpressed in tobacco,and transgenic lines(#8 and #18)and wild type(WT)were treated with low temperature at the same time.In terms of phenotype,the degree of leaf curl of transgenic tobacco was lighter than WT;In consideration of physiological indexes,the MDA content and electrolyte leakage of the transgenic lines were lower;What’s more,the leaf staining area of the transgenic line was smaller by DAB and NBT staining,indicating that the accumulation of ROS in the transgenic lines was fewer.The transgenic lines had stronger capacity of scavenging ROS,implying that PtrTZF1 positively regulate the cold resistance of plants.Through Agrobacterium tumefaciens-mediated transformation of citrus epicotyls,positive overexpression of lemon and trifoliate orange gene editing plants were obtained.3.After transient expression in tobacco,the strong transcriptional activation activity of PtrTZF1 was verified by GUS staining and LUC experiment.Therefore,PtrTZF1 is a transcription factor with transcriptional activation activity.In the yeast system,we found that the whole length of PtrTZF1 and its fragment PtrTZF1-D3(1～516 aa)had auto-activation activity,while the fragments PtrTZF1-D1(1～197 aa)and PtrTZF1-D2(1～416 aa)had no auto-activation activity,indicating that the auto-activation segment of PtrTZF1 was located at the C-terminal.4.Through yeast two-hybrid experiment,243 potential interacting proteins were screened.Firstly,we verified that 28 candidate proteins could interact with PtrTZF1,including Ptr ERD10,Ptr PR1,Ptr GRP and Ptr MYB44,etc.And then we used Bi FC experiment for further verification.In addition,we also analyzed the expression patterns of target proteins under low temperature,and found that they could be induced by low temperature.5.The transient expression experiment of sweet orange callus showed that the full length of PtrTZF1 promoter was induced by low temperature.In order to clarify the region of PtrTZF1 promoter induced by low temperature,we constructed different length deletion promoter vectors,and transformed into Arabidopsis thaliana by soaking flowers to obtain positive T1 generation seeds. |
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