Cloning And Expression Analysis Of Invertase Inhibitor Genes Of Day Lily (Hemerocallis Spp)

Invertase inhibitor is a low molecular weight protein（17～23k Da）,which can bind invertase to form an inactive complex and regulate invertase activity at the post-translational level.Studies have shown that the post-translational regulation of invertase is an effective way to reduce the invertase activity of transgenic plants and plays an important role in regulating plant growth and development.Inhibition of invertase inhibitor expression can increase invertase activity,delay leaf senescence,affect fruit and seed development and nutrients.In this study,petal-specific invertase inhibitors were cloned from daylily（Hemerocallis spp）.The function of aforementioned daylily invertase inhibitors genes was studied by genetic transformation,gene editing and bioinformatics analysis.At the same time,this work provides a basis for future study on the function of invertase inhibitor genes in regulating flower bud development and petal senescence.The main contents include following four parts.（1）According to the results of the third-generation transcriptome sequencing and the second-generation transcriptome of different organs of daylily,flower-specific invertase inhibitor genes were screened by local Blast.The full-length coding sequence（CDS）of invertase inhibitor were cloned for the first time from daylily by reverse transcription polymerase chain reaction（RT-PCR）.They were named Hf INH1,Hf INH2 and Hf INH3,respectively.The structure and function of these Hf INH proteins were predicted by bioinformatics analysis,and the subcellular localization of two members was studied by transient expression in tobacco leaves.In addition,the expression patterns in different tissues of daylily were analyzed by real-time fluorescent quantitative PCR,which verified the petal-specific expression characteristics of above three invertase inhibitors.（2）The over-expression vector p Cm PAP-Ubi-Hf INHs-NOS containing the above mentioned three invertase inhibitor genes were constructed.The tobacco genetic transformation was performed by Agrobacterium infection,and the hygromycin resistant transgenic tobacco was screened.The genomic DNA was used as a template for preliminary identification,and then verified by fluorescence quantitative PCR.（3）The genomic DNA sequence of the above three invertase inhibitors were analyzed by genomic PCR technology according to the coding sequence of invertase inhibitor gene information.The gene editing vector p Cas9PF-Hf INH1 were then generateded,and pollen tube pathway was used for daylily transformation.As a result,2427seeds of were harvested after 30 days,and 378 seedlings were obtained after sowing.Among them,6 plantlets were detected for containing Cas9 protein.However,the results here showed that no gene mutation was detected after sequencing.（4）p BI121 plasmid containing35S:GUS fusion gene was used in daylily pollen electroporation.The rate of daylily pollen germination and transformation under different electroporation conditions was investigated,and the optimal parameters of daylily pollen electroporation were selected,which provided a novel pathway for daylily genetic transformation and gene editing.The main conclusions of the experiment are as follows.1.The full-length CDS of three daylily invertase inhibitors were cloned.The CDS of Hf INH1 is 548 bp,encoding 177 amino acids.The CDS of Hf INH2 is 341 bp,encoding 112amino acids.The CDS of Hf INH3 is 577 bp,encoding 186 amino acids.Phylogenetic and molecular evolution analysis showed that the proteins encoded by these three genes contained four conserved cystine residues,which was the characteristics of all known plant invertase inhibitors.PCR amplification showed that the genomic DNA and c DNA sequences of INH genes were consistent completely,suggesting that there was no intron in the three Hf INH genes.2.In order to study the subcellular localization of these Hf INHs,the recombinant plasmids p131-Hf INH1-YFP、p131-Hf INH2-YFP and p131-Hf INH3-YFP were generated.After sequencing,the positive plasmids were extracted and used for Agrobacterium transformation.The leaf injection method was used to infect the lower epidermis of Nicotiana benthamiana leaves.After 3 days of culture,the fluorescence signals in tobacco epidermal cells were observed by Leica TCS SP8 laser confocal microscope.The results showed that Hf INH1 was located in the cell wall,Hf INH2 was located on the cell membrane and Hf INH3 fusion protein was expressed on the cell membrane and vacuole membrane.3.The expression patterns of aforementioned invertase inhibitor genes in different tissues of daylily were analyzed by q RT-PCR with different organs of‘Black Eyed Stella’as materials.The results showed that aforementioned invertase inhibitor had the highest expression level in petals of daylily,suggesting that these invertase inhibitor might be related to flower bud development and petal senescence of daylily.4.Plant overexpression vectors containing CDS of aforementioned invertase inhibitor genes were generated,and tobacco（N.benthamiana）genetic transformation was performed via Agrobacterium.The transgenic tobacco plants were screened by PCR amplification of genomic DNA.The expression level of Hf INHs in transgenic tobacco was detected by q RT-PCR.The results showed that the expression level of invertase inhibitor gene was significantly increased compared with that in the wild type tobacco plants.5.The mutation target sites were designed according to the specific cutting principle of CRISPR/Cas9 system,namely,PAM（NGG or CCN）.And the p Cas9PF-Hf INH1 gene editing vector was generated.Daylily genetic transformation was performed by pollen tube pathway,and Cas9 protein and g RNA were detected in 6 transgenic plants.However,the sequencing results using genomic DNA showed that there was no mutation happened.6.The pulse field strength and repeat times of pulses for daylily pollen electroporation were investigated using mature pollen of H.spp’Black Eyed Stella’.The effects of different pulse field strength and repeat times of pulses on pollen transformation rate were compared.The results here showed that the optimal parameters of pollen electroporation of daylily were1000 V·cm^-1for 3 times,which provided a reference for obtaining transgenic plants and their application in gene editing.