Cloning and temporal-spatial expression analysis of dfr gene from Scutellaria baicalensis with different colors / 生物工程学报

Dihydroflavanol-4-reductase (Dfr) is a key enzyme that regulates the synthesis of anthocyanin and proanthocyanidin in the flavonoid biosynthesis pathway. To investigate the difference of dfr gene in Scutellaria baicalensis Georgi with different colors in the same ecological environment, three complete full-length sequences of dfr gene were cloned from the cDNA of S. baicalensis with white, purple-red and purple colors using homologous cloning and RACE techniques. The three genes were named Sbdfr1, Sbdfr2 and Sbdfr3, respectively, and their corresponding structures were analyzed. The results showed that all three Dfr proteins have highly conserved NADPH binding sites and substrate-specific binding sites. Phylogenetic analysis showed that they are closely related to that of the known S. viscidula (ACV49882.1). Analysis of key structural domains and 3D models revealed differences in the catalytically active regions on the surface of all three Dfr proteins, and their unique structural characteristics may provide favorable conditions for studying the substrate specificity of different Dfr proteins. qRT-PCR analysis shows that dfr was expressed at different level in all tissues except the roots of S. baicalensis in full-bloom. During floral development, the expression level of dfr in white and purple-flowered Scutellaria showed an overall upward trend. In purple-red-flowered Scutellaria, the expression first slowly increased, followed by a decrease, and then rapidly increased to the maximum. This research provides a theoretical basis for further exploring the mechanism and function of Dfr substrate selectivity, and are of great scientific value for elucidating the molecular mechanism of floral color variation in S. baicalensis.

Construction and characterization of an infectious clone of Soybean mosaic virus isolate from Pinellia ternata / 生物工程学报

Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.

Molecular identification and sequence analysis of broad bean wilt virus 2 isolates from atractylodes macrocephala Koidz / 病毒学报

To identity the pathogen that causes the mosaic and yellowing symptoms on Atractylodes macrocephala Koidz in Jiangxian, Shanxi province, biological inoculation, sequence-independent amplification (SIA),RT-PCR and other identification methods were used. The results showed that the chlorotic and necrosis symptoms occurred in the indicator plant Chenopodium quinoa after it was infected with the pathogen,and the same symptoms appeared after the reinoculation of healthy Atractylodes macrocephala Koidz; this reflected that the disease was likely to be caused by a virus. The results of SIA and sequencing showed that Broad bean wilt virus 2 (BBWV2) was present in severely mosaic Atractylodes macrocephala Koidz leaves. To further characterize the BBWV2 isolate from Atractylodes macrocephala (BBWV2-Am), the polyprotein partial gene encoded by BBWV2-Am RNA2 was cloned and sequenced. Sequence alignments showed that the nucleotide sequence identity of BBWV2-Am SCP and LCP genes ranged from 79.3% to 87.2% and from 80.1% to 89.2% compared to other BBWV2 strains,respectively; the deduced amino acid sequence similarities of the two gene products ranged from 91.2% to 95.7% and from 89.44 to 95.5%, respectively,compared to those of other BBWV2 strains. Phylogenetic comparisons showed that BBWV2-Am was most likely to be related to BBWV2-Rg,but formed an independent branch. This is the first report of BBWV2 in Atractylodes macrocephala Koidz.