Gene Editing And Function Research Of The Genes Related To Controling The Formation Of Lateral Meristems(Las,Bl,Rev)in Nicotiana Tabacum

The ideal plant type is conducive to increasing crop yields.For example,fruit set percentage can be improved after tree pruning and the yield of rice and wheat will be improved by increasing the number of tillers.The morphology of the aerial parts of plants is mainly determined by the branch development.The branch development of plants is divided into three stages:the formation of lateral meristems is mainly regulated to genetic factors;the formation of axillary buds and the growth and dormancy of axillary buds are mainly determined by developmental process,external environment and some plant hormones such as auxin,cytokinin and strigolactone.Tobacco is an economic crop which harvested leaves as a raw material for production.It’s monopodial branching plant.The axillary bud of tobacco does not grow result from inhibiting by the apical dominance during vegetative growth phase.The apical advantage is weakened when vegetative growth stage of tobacco transformed to reproductive growth stage.The apical dominance rescission will lead to the concentration of auxin reduced and cytokinin increased in the leaf axil after decapitation.This will promote the growth of axillary buds and form new branches.The growth of axillary buds is not conducive to nutrients accumulation in leaves,which will reduce the commercial value of tobacco leaves.Tobacco topping is a time-consuming and laborious task.Based on this,the relevant genes(las,bl,rev)regulating lateral meristems in tobacco were knocked out by CRISPR/Cas9 gene editing method to obtain mutants which are axillary buds reduced after the topping.Gene function research will provide a theoretical basis for tobacco variety innovation.The main research results obtained are as follows:Nine HD-ZipIII transcription factor family members were identified from the tobacco genome by homology alignment,named NtHB1-NtHB9.NtHB6 and NtHB9 have the highest homology to rev,79.88%and 80.24%respectively.Ntlasl and Ntlas2,two homologs genes of las,were identified from the tobacco genome by homologous alignment and phylogenetic analysis.Ntraxl and Ntrax2 which is homologous genes of bl were downloaded from NCBI.The primers were designed to clone the CDS sequences of NtHB6/NtHB9,Ntlas1/Ntlas2 and Ntraxl/Ntrax2.According to gene structure and CDS sequence,nine edited target sites were designed.Ten single knockout vectors and double knockout vectors were constructed.Agrobacterium-mediated genetic transformation infected wild-type tobacco leaves and obtain regenerated plants.The results of mutation detection showed that we obtained mutants target for las,bl and rev,and mutant forms were deletion,insertion and substitution of bases.T1 generation mutants were obtained by bagging self-intersection.Nine single gene knockout mutant strains and three double gene knockout mutant strains were obtained successfully.The results of mutation analysis showed that rev2-4 and rev2-6 detected double genes mutation of NtHB6 and NtHB9.Rev2-6 homozygous mutant showed obvious developmental defect:the apical bud arrested at early and developed heteromorphic leaves.The shape of heteromorphic leaf was circular,trumpet-like and asymmetrically fan-shaped.The development of veins was also affected.The roots of the apical bud defect mutants could not develop.Rev1-3 detected a single gene mutation in NtHB9.Rev1-3 homozygous mutants grown like wild-type,and the number of axillary buds was not significantly different from wild type after decapitation 10 days,20 days and 30 days.Rev2-4 heterozygous grown like wild-type,but the average number of axillary buds was lower than wild type after decapitation 10 days,20 days and 30 days.The pleiotropic effects of NtHB6 and NtHB9 on tobacco development were analyzed by the study of mutant rev2-6.The effects include the following aspects:firstly,the effect on apical meristem.Apical bud could not develop and the expression of genes involving in apical meristem development changed significantly.Secondly,the effect of non-apical meristem.The surface of the heterotypic leaf grown convex tissue or frond.The thickness of the leaf was obviously increased,and the weight per unit leaf area was increased.The surface of rev2-6 leaves was observed by scanning electron microscopy.It was found that the number of glandular hairs was significantly reduced and the size of epidermal cells were significantly increased.Thirdly,the effects on leaf development include three aspects:the leaves could not establish normal leaf polarity and the expression of genes involving leaf polarity was changed significantly;The veins could not develop and vascular bundle became smaller;The content of total sugar,protein and chlorophyll decreased in leaves.