Establishment Of Genetic Transformation System Of Erigeron Breviscapus Plastid

Deng Zhan Xi Xin is Erigeron breviscapus（Vant.）Hand.-Mazz.of the family Asteraceae,which is a perennial herbal medicinal plant.This species has a short cultivation period and a high regeneration capacity,from seed germination to maturity in 30～40 d.New leaves can be grown in a relatively short time by regular succession,while maintaining roots and stems.Currently,a number of studies have established a mature and stable tissue culture system of E.breviscapus,and different organs and different parts of organs can be used as explant material for establishing the tissue culture system.In addition,the sequencing of the transcriptome and genome of E.breviscapus has been completed in recent years to further elucidate the genetic background of the species,and the above characteristics suggest that E.breviscapus has the potential to become a chassis plant.The establishment of plant genetic transformation system is the key to the development of chassis plants,which is important for the synthetic biology research of natural products（such as food nutrients or food additives,etc.）.Compared with the common nuclear genomic genetic transformation system,the plastid genomic genetic transformation system has the advantages of targeted integration,stable inheritance,and efficient expression.At present,the system has been established in common model plants and crops such as tobacco,Arabidopsis,lettuce and tomato,but the low transformation efficiency,difficulty of homogenization,and sterility of regenerated plants further limit the range of species to which the plastid genetic transformation system can be applied.To further expand the range of species to which plastid transformation can be applied and to lay the foundation for the development of E.breviscapus as a chassis plant,this study established a modified high-salt low-p H method for extraction of E.breviscapus chloroplast DNA（cp DNA）,and completed the sequencing and analysis of the E.breviscapus chloroplast genome;on this basis,the endogenous chloroplast expression regulatory elements of E.breviscapus were screened and identified,and the chloroplast expression vectors p Bt Ft（FPS）and p Bt FSt（FPS,SQE）for mogrol biosynthesis were constructed using the above elements and enzyme genes related to the biosynthetic pathway of mogrol;the critical selection pressure for the selective culture of transplatomic cells was determined by the spectinomycin sensitivity assay.The gene gun bombardment transformation conditions were optimized by orthogonal experiments using E.breviscapus leaves as explants,and the transplastomic plants were obtained,thus establishing the plastid genetic transformation system of E.breviscapus.The results of this study laid the foundation for the subsequent biosynthesis of natural products（food nutrients or food additives）by using E.breviscapus,and also provided reference for the establishment of plastid genetic transformation system in other plants,the main results are as follows:1.Establishment of the cp DNA extraction method of E.breviscapusUsing mature leaves of E.breviscapus as materials,the effects of high-salt method,Na OH low-salt method and modified high-salt low-p H method on chloroplast isolation and cp DNA extraction were compared.When the chloroplasts isolated by the above three methods were observed by fluorescence microscope,it can be seen in the natural light field that the chloroplasts separated by the modified high-salt low-p H method and Na OH low-salt method are round or oval and uniformly dispersed,with few cell fragments and clear background.Chloroplasts separated by high-salt method are in large number and more dense,with more cell fragments and serious background pollution;In the dark field,it can be seen that the location of the spontaneous red fluorescence of chloroplasts separated by the modified high-salt low-p H method and Na OH low-salt method is consistent with the location of the chloroplasts in the natural light field.The spontaneous red fluorescence of chloroplasts separated by the high-salt method cannot be observed at most of the corresponding positions.The above results show that,the modified high-salt low-p H method and the Na OH low-salt method can effectively separate the chloroplasts from the leaves of E.breviscapus and maintain the integrity of the chloroplast structure.Chloroplasts separated by the high-salt method have an incomplete structure and severe background contamination;the chloroplasts obtained by the above three methods were completely lysed,and cp DNA was extracted.The concentration of cp DNA extracted by the modified high-salt low-p H method was329 ng/u L.The value of A_{260 nm}/A_{280 nm} is 1.99,and the value of A_{260 nm}/A_{230 nm} is 2.23.The concentration of cp DNA extracted by the high-salt method is 168.4 ng/μL.The value of A_{260 nm}/A_{280 nm} is 1.47,the value of A_{260 nm}/A_{230 nm} is 0.95,and the concentration of cp DNA extracted by the Na OH low-salt method is 88.0 ng/μL.The value of A_{260 nm}/A_{280 nm} is 1.43,and the value of A_260nm/A_{230 nm} is 1.38.The above results show that the cp DNA extracted by the modified high-salt low-p H method has the highest yield,and the content of impurities such as protein,phenols and polysaccharides is low.The yields of cp DNA extracted by the high-salt method and the Na OH low-salt method are lower than those of cp DNA extracted by the modified high-salt low-p H method and contain impurities such as protein and phenols;the integrity of the cp DNA was detected by gel electrophoresis,and the results showed that only the cp DNA extracted by the modified high-salt low-p H method had clear and bright bands without any obvious trailing,indicating that the cp DNA extracted by this method had high purity and good integrity without any obvious degradation.2.Sequencing and analysis of the chloroplast genome of E.breviscapusThe assembly of the chloroplast genome of E.breviscapus was completed by data filtering,splicing and correction of the raw sequencing data.The chloroplast genome of E.breviscapus is152,164 bp in size with a GC content of 37.2%and consists structurally of two inverted repeat regions（IR,24,699 bp）,a large single copy region（LSC,84,657 bp）and a small single copy region（SSC,18,109 bp）.Gene function annotation and structure prediction of the chloroplast genome of E.breviscapus using CPGAVAS2 resulted in the annotation of 127 coding genes,including 83protein-coding genes,36 transfer RNA（t RNA）genes and 8 ribosomal RNA（r RNA）genes.Among them,95 genes were single-copy genes and 16 genes were located in two inverted repeat regions,including 7 t RNA,4 r RNA and 5 protein-coding genes;the codon usage bias of the E.breviscapus chloroplast genome was analyzed using Codon W,and the results showed that there were 31 codons with relative synonymous codon usage（RSCU）≥1.There were 12 codons ending in A（UUA,AGA,CAA,GGA,etc.）,16 codons ending in U（UCU,GCU,ACU,UAU,etc.）,and three codons ending in G（UUG,UGG and AUG）.It shows that codons ending in A and U appear more frequently in the chloroplast genome of E.breviscapus and are preferred codons,while codons ending in C and G are non-preferred codons;Simple sequence repeats（SSRs）and long repetitive sequences in the chloroplast genome of E.breviscapus were identified using MISA and vmatch.A total of 223 SSRs were annotated,including 142 complex repeat type SSRs,50 dinucleotide SSRs,12 trinucleotide SSRs,14 tetranucleotide SSRs,four pentanucleotide SSRs,and one hexanucleotide SSR;in addition,there are three unique long repetitive sequences in the chloroplast genome of E.breviscapus,including two palindromic repeats and one forward repeat,the size of the two palindromic repeats are 48 bp and 24,699 bp,respectively,and the size of the forward repeat is 39 bp.3.Screening,functional identification and application of chloroplast regulatory elements from E.breviscapusTwo homologous recombination fragments,two promoters,two 5′UTR sequences,three 3′UTR sequences,one SD sequence and one intercistronic expression element（IEE）were preliminarily identified from the chloroplast genome of E.breviscapus by bioinformatic analysis.Using E.breviscapus cp DNA as a template,the trn I gene,the trn A gene,and partial 5′and 3′flanking sequences of both genes were amplified by PCR as homologous recombinant fragments of the chloroplast expression vector.The endogenous promoter of psb A gene and 16S r DNA gene,the endogenous 3′UTR sequences of psb A,rbc L gene and rps16 gene,and an IEE derived from the psb N-psb H intergenic region were amplified by PCR as expression regulatory elements for the construction of chloroplast expression vectors.The above homologous recombinant fragment,the expression regulatory element,the reporter gene EGFP,the selective marker gene aad A and the cloning vector p Bluescript SK（+）were used to construct the chloroplast fluorescent protein expression vector of E.breviscapus.The expression vector was then transformed into E.coli,and the prokaryotic expression characteristics of the expression regulatory element were identified by spectinomycin resistance test and fluorescence intensity measurement.All E.coli strains transformed with chloroplast expression vectors were resistant to spectinomycin and capable of expressing enhanced green fluorescent protein（EGFP）,and the results showed that the chloroplast expression regulatory elements obtained by screening and cloning had the correct biological functions and could be used for the establishment of the subsequent plastid genetic transformation system of E.breviscapus;based on the optimization and synthesis of the key enzyme genes（FPS,SQE,CDS,EPH,CYP87D18,CPR）related to the mogrol biosynthesis pathway according to the codon usage bias of the E.breviscapus chloroplast genome,the chloroplast expression regulatory elements and pathway-related enzyme genes were used to construct the vectors p Bt Ft（FPS）and p Bt FSt（FPS,SQE）for chloroplast expression of mogrol biosynthesis with reference to the structure of the chloroplast operon.4.Establishment of chloroplast genetic transformation system of E.breviscapusThe critical selection pressure of spectinomycin in the leaves of E.breviscapus was determined by the spectinomycin sensitivity test.The results showed that when the final concentration of spectinomycin was 40 mg/L,it could effectively inhibit the growth of untransformed callus and completely whiten the untransformed callus cells;the gene gun bombardment transformation conditions were optimized by orthogonal experiments,and the number of factors studied was 5（helium pressure,target distance,chamber vacuum pressure,number of bombardments,plasmid DNA concentration）,and the number of levels of each factor was 3.The gene gun transformation experiments were designed with reference to the orthogonal table of L₁₈（3⁷）;the leaves of sterile seedlings of E.breviscapus were used as receptors,p Bt Eat3 vector was used as donor,transformation experiments were mediated by gene gun,and transplastomic plants were obtained by multiple rounds of selective culture.The results showed that the optimal transformation conditions for gene gun bombardment are 1100 psi helium pressure,9 cm target distance,26mm Hg chamber vacuum pressure,1 bombardment number and 1μg/μL plasmid DNA concentration;PCR identification results showed that using the cp DNA of the transgenic plants as a template,specific primers were used to amplify EGFP and aad A genes,and electrophoretic detection showed DNA bands matching the size of EGFP and aad A gene sequence fragments,indicating that the above genes were successfully integrated into the chloroplast genome of E.breviscapus.The LF-EGFP and RF-aad A sequences were amplified using specific primers,and DNA bands matching the size of the LF-EGFP and RF-aad A sequences were detected by gel electrophoresis,indicating that the EGFP and aad A gene expression cassettes in the p Bt Eat3 vector were targeted and integrated into the chloroplast genome of E.breviscapus;the chloroplasts of transplastomic and wild-type plants were observed by laser confocal microscopy.In the natural light field,the chloroplast structure of transplastomic plants and wild-type plants was intact with no obvious difference;in the dark field,spontaneous red fluorescence could be observed from the chloroplasts of transplastomic plants and wild-type plants,while no green excitation fluorescence could be observed from wild-type plants and could be observed from transplastomic plants,indicating that the EGFP gene has been integrated into the chloroplast genome of E.breviscapus and is correctly expressed.