Cloning, Characterization, Spatio-Temporal Expression Analysis Of 4-Coumarate:Coa Ligase (4CL) Genes And Genetic Transformation Of Sweet Sorghum (Sorghum Bicolor)

4-coumarate:CoA ligase (4CL, EC6.2.1.12) protein is the key rate-limiting enzyme in lignin biosynthesis. By homology analyzing the sequences, which have been identified the true 4CL genes of arabidopsis, rice, corn and poplar by blast in NCBI database, seven putative 4CL-like genes were identified in sorghum genome.The coding sequences of these seven 4CL-like genes were cloned by using the cDNA from the sweet sorghum variety’Da Li Shi’as the template. The amino acid sequences of these 4CL proteins in sweet sorghum are of about 90%similarity with corresponding 4CL proteins in rice. These 4CL proteins in sweet sorghum have been divided into two sub-categories:Class I (Sb07G007810, Sb04G005210, Sbl0G026130, Sb07G022040, Sb03G000610 and SbO6GO1663O) and Class Ⅱ (Sb04G031010).Seven 4CL-like gene from the sweet sorghum were constructed into the prokaryotic expression vector pET-28a(+)and pET-51b(+). The recombinant plasmid was introduced into E. coli BL21 cells for protein expression. Soluble protein was extracted by sonication. The expressed recombinant protein, which contained an N-terminal 6 His tag, was captured on a nickel resin column and eluted using imidazole. Purified protien were used for 4CL enzyme activity test. Enzyme activity assays verified that these seven 4CL proteins all had 4CL catalytic activities though with different substrate affinities, indicating these seven 4CL-like genes really belong to 4CL family. Sb07G007810, Sb04G005210, Sbl0G026130 and Sb07G022040, which belong to 4CL Class Ⅰ protein, showed high activity against the substrate 4-coumarate, cinnamate, caffeate and ferulate. Sb04G031010, which belongs to 4CL Class Ⅱ protein, showed relatively lower activity against the same substrates. Sb03G000610 and Sb06G016630, which belong 4CL Class Ⅰ protein, showed the lowest activity against the same substrates. None proteins displayed 4CL activity against sinapate substrate.By analysis the expression pattern of these seven 4CL genes in sweet sorghum, we figure out that in the roots Sb04G005210 had the highest expression and Sb07G007810 was the second. The two genes accounted for 93%～95% expression in the total expression amount of 4CL Class Ⅰ genes. In sweet sorghum leaves, Sb07G007810 had the highest expression and Sb4G005210 was the second. The two genes accounted for 90% in the total expression amount of 4CL Class Ⅰ genes. In sweet sorghum stems, Sb4G005210 had the highest expression. It accounted for 83% to 89% in the total amount of 4CL Class Ⅰ genes in seedling xylem. The 4CL Class Ⅰ genes, which involved in lignin biosynthesis, highly expressed in fast-growing stages. The expression level of the 4CL Class Ⅱ gene which participated in flavonoid synthesis was significantly increased in the parts which directly received solar radiation.Callus induction of sweet sorghum is the base of transgenic sweet sorghum research. In this study, we induced the sweet sorghum callus production by using different induction media and different parts of sweet sorghum. As a result, we found that the optimal medium for callus induction of mature embryo (seed) was modified MS basal culture medium+5 mg·L-1 2,4-D. The optimal medium for callus induction of plumulewas modified MS basal culture medium+3 mg·L-12,4-D. The optimal medium for callus induction of radiclewas modified MS basal culture medium+3 mg·L-1 2,4-D+0.5 mg·L-1 KT. The callus induction rate of young panicle was highest, and the optimal medium for callus induction of young panicle was modified MS basal culture medium+3 mg·L-1 2,4-D. Callus induced by young panicle had the highest differentiation rate. In conclusion, young panicle was the best explant to induce embryogenic callus, radicleplumuleAt present, manipulating the lignin biosynthesis by genetic engineering is a hot point in sweet sorghum research. In order to obtain reduced lignin content sweet sorghum, we constructed the interference vector pFGC5941-4CL-RNAi specific for sweet sorghum 4CL genes. We transformed pFGC5941-4CL-RNAi into callus of sweet sorghum by Agrobacterium-mediated transformation and gene gun transformation.