Optimization Of Citrus Protoplast Transient Transformation System And Using CRISPR/cas9 Genome Editing System To Modify Gene In Fortuella Hindsii

Citrus is the world’s highest yielding fruit.Due to the characteristics of long childhood,multiple embryos,low fertility of some important commercial breeds and even infertility,the use of traditional breeding methods to improve citrus progress is slow.CRISPR/Cas9 system as a rapid development of genome editing technology in recent years can accurately and effectively induce specific mutations,specifically transform the genome,has become an important technology of plant gene function research and crop genetic improvement.The plants obtained by the transient transformation of plant protoplasts of the CRISPR/Cas9 system are non-transgenic germplasm resources.The long progeny screening step can be omitted,and the chimera is easily obtained by transforming the citrus epicotyl mediated by Agrobacterium.The protoplast transient transformation of the CRISPR/Cas9 system is a genetic editing of a single cell,and the probability of homozygosity is greatly increased.Since the transient transformation system of citrus protoplasts is not muture,this study aims to optimize the transient transformation system of citrus protoplasts and improve the transient transformation efficiency of citrus mesophyll protoplasts to fully utilize subcellular localization and BiFC experiments for the function of citrus species genes.Improve the transient transformation efficiency of citrus calli protoplasts to create new citrus germplasm by using the CRISPR/Cas9 system;Introduce three types of different CRISPR/Cas9 vector construction systems;and use Agrobacterium-mediated genetic transformation of the epicotyls of Fortuella hindsii.The CsP5CS2 gene of interest was knocked out and positive plants were obtained for the later study of gene function.The experimental results include the following aspects:1.Optimize the transient transformation system of citrus protoplasts.1)vacuum penetration enzymatic hydrolysis method to shorten the enzymolysis time,the separation and purification of protoplasts sandwich method,remove the broken protoplast impurities and cell clusters that have not been completely enzymatic hydrolysis,greatly improve the integrity and viability of the resulting protoplasts exceeds 90%;2)The co-transformation process during the transient transformation of citrus protoplasts is changed and co-transformed with a 37℃water bath for 30 min,making the transient transformation efficiency of citrus mesophyll protoplasts 19.10%significant improved to 42.04%.（3）The concentration of Ca²⁺in PEG solution increased to 30 mmol/L.Co-transformation treatment at 37℃water bath for 30 min resulted in a significant increase in the transient transformation efficiency of citrus protoplasts from 3.63%to 13.18%.2.Application of Transient Transformation System of Citrus Protoplasts.Subcellular localization experiments were performed using transient transformation of citrus protoplasts.The protein encoded by CsSPL3 was localized in the protoplast cell nuclei of citrus callus.BiFC experiments using protoplast transformation of citrus mesophyll were performed.The results showed that CsSPL14 interacts with CsARK1 protein.3.CRISPR/Cas9 genome editing system to modify CsP5CS2 gene in Fortuella hindsii.The gene CsP5CS2,which encodes a rate-limiting enzyme for proline synthesis in citrus,was used as the target gene.Two gRNAs were designed based on the target site using the CRISPR-P website to construct the vector pKSE-GFP-CsP5CS2.Agrobacterium-mediated genetic transformation of the epicotyl of Fortuella hindsii was used to obtain transgenic positive buds and plants,and sequencing the DNA of transgenic plants.The results show that the CRISPR/Cas9 system has an editing effect on the target gene CsP5CS2 of Fortuella hindsii and has a variety of editing effects:2 bp,8 bp,50 bp deletion and 1 bp insertion at 3-4 bases before the PAM locus.The positive plants obtained in this experiment can be used for subsequent studies of CsP5CS2 gene function.