Functional Characterization of the MeSSIII-1 Gene and Its Promoter from Cassava.

Soluble starch synthases (SSs) play important roles in the synthesis of cassava starch. However, the expression characteristics of the cassava SSs genes have not been elucidated. In this study, the MeSSIII-1 gene and its promoter, from SC8 cassava cultivars, were respectively isolated by PCR amplification. MeSSIII-1 protein was localized to the chloroplasts. qRT-PCR analysis revealed that the MeSSIII-1 gene was expressed in almost all tissues tested, and the expression in mature leaves was 18.9 times more than that in tuber roots. MeSSIII-1 expression was induced by methyljasmonate (MeJA), abscisic acid (ABA), and ethylene (ET) hormones in cassava. MeSSIII-1 expression patterns were further confirmed in proMeSSIII-1 transgenic cassava. The promoter deletion analysis showed that the -264 bp to -1 bp MeSSIII-1 promoter has basal activity. The range from -1228 bp to -987 bp and -488 bp to -264 bp significantly enhance promoter activity. The regions from -987 bp to -747 bp and -747 bp to -488 bp have repressive activity. These findings will provide an important reference for research on the potential function and transcriptional regulation mechanisms of the MeSSIII-1 gene and for further in-depth exploration of the regulatory network of its internal functional elements.

A Transformation and Genome Editing System for Cassava Cultivar SC8.

Cassava starch is a widely used raw material for industrial production. South Chinese cassava cultivar 8 (Manihot esculenta Crantz cv. SC8) is one of the main locally planted cultivars. In this study, an efficient transformation system for cassava SC8 mediated with Agrobacterium strain LBA4404 was presented for the first time. Cassava friable embryogenic calli (FECs) were transformed through the binary vector pCAMBIA1304 harboring GUS- and GFP-fused genes driven by the CaMV35S promoter. The transformation efficiency was increased in the conditions of Agrobacterium strain cell infection density (OD600 = 0.65), 250 µM acetosyringone induction, and agro-cultivation with wet FECs for 3 days in dark. Based on the optimized transformation protocol, approximately 120-140 independent transgenic lines per mL settled cell volume (SCV) of FECs were created by gene transformation in approximately 5 months, and 45.83% homozygous mono-allelic mutations of the MePDS gene with a YAO promoter-driven CRISPR/Cas9 system were generated. This study will open a more functional avenue for the genetic improvement of cassava SC8.

MeNINV1: An Alkaline/Neutral Invertase Gene of Manihot esculenta, Enhanced Sucrose Catabolism and Promoted Plant Vegetative Growth in Transgenic Arabidopsis.

Alkaline/neutral invertase (A/N-INV) is an invertase that irreversibly decomposes sucrose into fructose as well as glucose and plays a role in plant growth and development, starch synthesis, abiotic stress, and other plant-life activities. Cassava is an economically important starch crop in tropical regions. During the development of cassava tuber roots, A/N-INV activity is relatively high, which indicates that it may participate in sucrose metabolism and starch synthesis. In this study, MeNINV1 was confirmed to function as invertase to catalyze sucrose decomposition in yeast. The optimal enzymatic properties of MeNINV1 were a pH of 6.5, a reaction temperature of 40 °C, and sucrose as its specific catalytic substrate. VB6, Zn2+, and Pb2+ at low concentrations as well as EDTA, DTT, Tris, Mg2+, and fructose inhibited A/N-INV enzymic activity. In cassava, the MeNINV1 gene was mainly expressed in the fibrous roots and the tuber root phloem, and its expression decreased as the tuber root grew. MeNINV1 was confirmed to localize in chloroplasts. In Arabidopsis, MeNINV1-overexpressing Arabidopsis had higher A/N-INV activity, and the increased glucose, fructose, and starch content in the leaves promoted plant growth and delayed flowering time but did not change its resistance to abiotic stress. Our results provide new insights into the biological function of MeNINV1.

Clinical features and prognostic influences of retropharyngeal lymph node metastasis in oropharyngeal and hypopharyngeal squamous cell carcinoma: a meta analysis / 中华耳鼻咽喉头颈外科杂志

Objective: To compare the clinical features and prognoses of patients with oropharyngeal or hypopharyngeal squamous cell carcinoma (SCC) with and without retropharyngeal lymph node metastases. Methods: PubMed, Cochrane, Web of Science, CNKI, VIP and Wanfang databases were searched for published literatures on retropharyngeal lymph node metastasis of oropharyngeal or hypopharyngeal squamous cell carcinoma (1900, 2021), and outcome indicators such as survival rate and related clinical features were extracted. The quality evaluation of the included literatures was carried out. RevMan 5.4 and Stata 16.0 software were used for data analysis. Results: A total of 18 literatures were included. Meta analysis showed that 3-year and 5-year survival rates and 5-year disease-specific survival rate of patients with oropharyngeal or hypopharyngeal squamous cell carcinoma with retropharyngeal lymph node metastases were lower than those without metastases, 46.1% vs. 53.0%, 40.8% vs. 62.5% and 35.9% vs. 53.1%, respectively, and the differences were statistically significant (OR values were 0.26, 0.38, 0.38, and 95%CI were 0.10-0.69, 0.28-0.51, 0.23-0.65, respectively, all P values<0.05). There were statistically significant differences in clinical stage (III-IV), T stage (T3+T4), N stage (N2), positive cervical lymph node metastases and number of lymph node metastases (≥3) between the two groups (OR values were 4.28, 2.20, 2.88, 10.83, 6.53, and 95%CI were 1.70-10.74, 1.35-3.58, 1.90-4.34, 3.57-32.95, 1.75-24.38, respectively, all P<0.05). The sensitivity and specificity of preoperative imaging for diagnosing retropharyngeal lymph nodes metastases were respectively 0.72 (95%CI=0.54-0.85) and 0.98 (95%CI=0.74-1.00), and the area under curve (AUC) of summary receiver operating characteristic curve (SROC) was 0.84 (95%CI=0.80-0.87). Conclusions: The survival rate of patients with oropharyngeal or hypopharyngeal squamous cell carcinoma with retropharyngeal lymph node metastasis is significantly reduced, the clinical stage and T stage are late, and the cervical lymph node metastasis rate is high. Retropharyngeal lymph node metastasis is more insidious, the sensitivity of preoperative imaging diagnosis is not high.