Transcriptome Profiling of Barley Cultivar Hua 30 MDEC in Response to <i>Agrobacterium</i> Infection

Agrobacterium-mediated transformation has been widely used in plants. However, the mechanism in plant cells’ response to Agrobacterium infection was very complex. The mechanism of the determinants in host cell remains obscure, especially in barley, which is recalcitrant for Agrobacterium-mediated transformation. In the present study, microspore-derived embryogenic calli (MDEC) from barley elite cultivar were employed as unique subjects to characterize the mechanisms during the Agrobacterium infection process. Hua 30 MDEC can be successfully infected by Agrobacterium. RNA-sequencing at different infection points (0, 2, 6, 12, 24 hpi) was performed. The average expressional intensity of the whole genomics increased from 0 to 2 hpi, and then decreased subsequently. More upregulated than downregulated differentially expressed genes (DEGs) were counted at the same time. GO enrichment analysis showed that protein modification was significantly overrepresented in upregulated DEGs. Chromosome-related biological processes, gene expression and cellular metabolic processes were significantly overrepresented in downregulated DEGs. KEGG analysis showed that plant defense responses, phenylpropanoid biosynthesis and biosynthesis of amino acids were significantly enriched across the infection time course. Nine DEGs related to defense responses were identified. All DEGs were upregulated from 2 to 24 hpi. We speculate that these genes are possibly related to Agrobacterium infection. These findings will provide deep insights into the molecular events occurring during the process of Agrobacterium-mediated transformation.     

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to <i>Sclerotinia sclerotiorum</i> in <i>Brassica napus</i>

Invertase (INV), a key enzyme in sucrose metabolism, irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose, thus playing important roles in plant growth, development, and biotic and abiotic stress responses. In this study, we identified 27 members of the BnaINV family in Brassica napus. We constructed a phylogenetic tree of the family and predicted the gene structures, conserved motifs, cis-acting elements in promoters, physicochemical properties of encoded proteins, and chromosomal distribution of the BnaINVs. We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B. napus cultivar Zhongshuang 11 using qRT-PCR. In addition, we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum. We used WGCNA (weighted coexpression network analysis) to uncover BnaINVregulatory networks. Finally, we explored the expression patterns of several BnaINV genes in cultivars with long (Zhongshuang 4) and short (Ningyou 12) siliques. Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens. Our findings could facilitate the breeding of high-yielding B. napus cultivars with strong disease resistance.     

基于转录组测序的细风轮花青素合成途径及关键酶基因分析

为进一步理解细风轮花青素合成途径,本研究利用华大基因BGISEQ-500平台对细风轮中的根、茎、叶、花4个组织进行了转录组测序,从头组装后得到128 856个Unigene。KEGG通路表明有40个Unigene编码了细风轮的花青素生物合成途径中6个关键酶。我们对其中的关键酶DFR（二氢黄酮醇还原酶）进行同源比对和空间结构模拟,结果显示DFR序列和结构均具有良好的保守性,且具有高度保守的NAD⁺结合位点,其二级结构主要由α螺旋和β折叠组成,在空间上α螺旋包裹着β折叠,形成“夹心饼干”样结构。     

<i>VvAGAMOUS</i> Affect Development of Four Different Grape Species Ovary

Grape pistil has an important influence on fruit size and quality. However, there were few studies on grape ovary, and the development process of the ovary is still unclear. Therefore, in this paper, four different grape varieties with different lengths of small inflorescences, namely ‘Musct Hambourg’ grape (Vitis vinifera), ‘Concord’ grape (Vitis labrusca), ‘ShanPuTao’ grape (Vitis amurensis) and ‘GongNiang2Hao’ grape (Vitis amurensis × Vitis vinifera) were used as test materials. Four varieties ovary were significant differences by means of stereomicroscope, paraffin section. The expression of ovary determining gene VvAGAMOUS (VvAG) and its development related genes VvCRABS CLAW (VvCRC) andVvAGAMOUS-LIKE 11 (VvAGL11) with similar functions during the development of different grape varieties were preliminarily explored using fluorescence quantitative test. The relationship between VvAG and VvCRC, VvAG and VvAGL11 were analyzed using Y1H assay. Our results showed that there were obvious abdominal sutures on the surface of expect for ‘Musct Hambourg’ grape, and existing poly carpels. The ovary development of ‘ShanPuTao’ and ‘GongNiang2Hao’ grape was completed when the inflorescence length was less than 1 cm, while the ‘Concord’ and ‘Musct Hambourg’ grape were fully developed when the length of inflorescence was 3–4 and 4–5 cm, respectively. VvAG and VvCRC began to express in large quantities after the formation of stamen primordia, while VvAGL11 during the forming of ovule primordia. Therefore, VvAG and VvCRC mainly regulated the development of stamens and carpels and also promote the development of ovules, while VvAGL11 major regulated the development of ovules. The promoters of VvCRC and VvAGL11 were bound by VvAG. This study provides an important theoretical basis for further research on the molecular mechanism of grape ovary development.     

Genome-Wide Analysis of the <i>KANADI</i> Gene Family and Its Expression Patterns under Different Nitrogen Concentrations Treatments in <i>Populus trichocarpa</i>

KANADI (KAN) is a plant-specific gene that controlled the polarity development of lateral organs. It mainly acted on the abaxial characteristics of plants to make the lateral organs asymmetrical. However, it had been less identified in woody plants. In this study, the members of the KAN gene family in Populus trichocarpa were identified and analyzed using the bioinformatics method. The results showed that a total of 8 KAN family members were screened out, and each member contained the unique GARP domain and conserved region of the family proteins. Phylogenetic analysis and their gene structures revealed that all KAN genes from P. trichocarpa, Arabidopsis thaliana, and Nicotiana benthamiana could be divided into four subgroups, while the eight genes in P. trichocarpa were classified into three subgroups, respectively. The analysis of tissue-specific expression indicated that PtKAN1 was highly expressed in young leaves, PtKAN6 was highly expressed in young leaves and mature leaves, PtKAN2, PtKAN5, and PtKAN7 were highly expressed in nodes and internodes, PtKAN8 was highly expressed in roots, and PtKAN3 and PtKAN4 showed low expression levels in all tissues. Among them, PtKAN2 and PtKAN6, and PtKAN4 and PtKAN5 might have functional redundancy. Under high nitrogen concentrations, PtKAN2 and PtKAN8 were highly expressed in mature stems and leaves, respectively, while PtKAN4, PtKAN5, and PtKAN7 were highly expressed in roots. This study laid a theoretical foundation for further study of the KAN gene-mediated nitrogen effect on root development.     

紫背天葵叶和花中花青素合成相关转录组基因分析

为获取紫背天葵(Gynura bicolor D C.)花青素合成代谢相关调控基因信息,该试验以紫背天葵叶片为材料,以其花朵为对照,进行转录组测序,并进行CHS、CHI、F3H等8类合成酶基因以及MYB、bHLH及WD40等3类转录因子检索,从中选取8个相关差异表达显著调控基因进行qRT-PCR验证分析。结果显示:(1)在紫背天葵中共获得72个花青素合成酶信息,其中差异表达明显的有1个F3′H和2个3GT下调,9个F3H基因中有上调基因4个和下调基因5个。(2)在紫背天葵中获取到238个MYB、113个bHLH和219个WD40转录因子,这3类转录因子中差异表达明显的分别为22个、16个和7个。(3)qRT-PCR结果显示,所选取的8个花青素合成相关调控基因,在紫背天葵叶及花朵中的下调表达趋势与转录组测序结果完全一致,但不同基因差异表达趋势略有不同。研究表明,在紫背天葵叶片和花朵中所存在的大量花青素合成代谢调控基因中,只有少量差异表达显著,但转录因子相比合成酶的调控更为复杂。     

Bioinformatics Analysis of Disease Resistance Gene <i>PR1</i> and Its Genetic Transformation in Soybeans and Cultivation of Multi-resistant Materials

In agricultural production, a single insect-resistant and disease-resistant variety can no longer meet the demand. In this study, the expression vector pCAMBIA-3301-PR1 containing the disease-resistant gene PR1 was constructed by means of genetic engineering, and the PR1 gene was genetically transformed to contain the PR1 gene through the pollen tube method. In CryAb-8Like transgenic high-generation T₇ receptor soybean, a new material that is resistant to insects and diseases is obtained. For T₂ transformed plants, routine PCR detection, Southern Blot hybridization, fluorescence quantitative PCR detection, indoor and outdoor pest resistance identification and indoor disease resistance identification were performed. The results showed that there were 9 positive plants in the routine PCR test of T₂ generation. In Southern Blot hybridization, both PR1 and CryAb-8Like genes are integrated in soybeans in the form of single copies. Fluorescence quantitative PCR showed that the expression levels of PR1 and CryAb-8Like genes are different in different tissues. The average expression levels of PR1 gene in plant roots, stems, and leaves are 2.88, 1.54, and 5.26, respectively. CryAb-8Like genes are found in roots, stems, and leaves. The average expression levels were 1.36, 1.39, and 4.25, respectively. The insectivorous rate of the CryAb-8Like gene in outdoor plants with positive insect resistance identification was 3.78%. The disc partition method was used indoors for pest resistance identification, and the bud length of transformed plants increased significantly. The average mortality rate of untransformed plants in indoor disease resistance identification was as high as 56.66%, and the average mortality rate of plants transformed with PR1 gene was 10.00%, and disease resistance was significantly improved. Therefore, a new material with resistance to diseases and insects is obtained.