Expression Analysis Of Two ATG8 Homologous Genes And In Vitro Callus Culture,Rapid Propagation Of Euphorbia Kansui

Euphorbia kansui Liou is a perennial herb of Euphorbia belongs to Euphorbiaceae,and it is a traditional medicine which is unique to China.The root is the effective medicinal part,which was used to cure edema,cough,and tumour and so on.E.kansui seedlings grow from the seeds or underground roots in July or August and stop the growth in December to complete the vegetative growth,to the next year,seedlings emerge in March,blossom in April,and fruit mature at the end of May to complete the reproductive growth.The whole plant of E.Kansui contains latex which is the cytoplasm of non-articulated branched laticifers.The effective ingredients such as terpenoids,flavonoids and other substances are produced and stored in latex.Therefore,there are both important theoretical and practical significance in researching development of laticifers.Autophagy is a highly conserved process of material cycling,which is mainly responsible for the degradation of long-lived proteins and organelles.Our research group had observed the phenomenon of fusion of small acidic organelles resembling lysosomes with organelles such as mitochondrion and plastid in laticifers of E.kansui.In addition,we had found some autophagy related genes in the transcriptome data.As a result,according to the transcriptome information,we cloned CDS sequences of two homologous genes of autophagy related protein ATG8 family?ATG8c and ATG8f?and analysed their expression pattern in root,stem,leaf and latex;and established the best conditions for rapid propagation of buds and root and callus induction,which lays the foundation for in-depth researches on autophagy model,and construction of transgenic ATG8 system to find the autophagy mechanism in laticifers of E.kansui.The results are as follows:?1?The CDS of ATG8 c is 381 bp,encoding 126 amino acids,and the predicting molecular weight of the protein is 14.53 kDa.The theoretical isoelectric point?pI?is 7.89;ATG8f CDS is 369 bp,encoding 122 amino acids,the predicting molecular weight is 14.08 kDa.The pI is 7.89.Homologous alignment and phylogenetic tree proved that two genes are highly conserved in evolution.Expression pattern analysis showed that the expression of ATG8 f was highest in leaf,ATG8 c was highest in latex,and ATG8 f was higher than that of ATG8 c in root,stem,leaf and latex.?2?The disinfection process of explants:cleaned stems or leaves which were collected from Botanical Garden,soaked these in 70%ethanol for 10 s,washed 2 times with sterile water,and then sterilized with 0.08%HgCl₂:leaves for 5 min,and stems for 8 min,washed with sterile water for 5 times at last.?3?The medium MS+6-BA 2.5 mg/L+2,4-D 2.0 mg/L was used to induct callus from the stems?without axillary buds?and leaves with inducing rates of 60.88%and 45.33%,respectively.The young stem segments with axillary buds were used as explants to induce multiple buds,and the medium was MS+6-BA 1.0 mg/L+IAA 3.0 mg/L with the inducing rate of 89.46%and proliferation rate of 8.93.The medium MS+6-BA 1.0 mg/L+NAA 0.2 mg/L was used to strengthen plantlet and the inducing rate were 89.39%.At last,the thick plantlet was inducted in medium of 1/2MS+IBA 0.5 mg/L?with rooting rate of 28.15%and the proliferation rate of 3.75?or 1/2MS+IBA 1.0 mg/L to generate roots?with rooting rate of 35.01%and the proliferation rate of 1.53?.