Exploring the basis of 2-propenyl and 3-butenyl glucosinolate synthesis by QTL mapping and RNA-sequencing in Brassica juncea

AbstractBrassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. These RNA-Seq results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea.

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Compatative Transcriptome Analysis Reveals Sesquiterpene Biosynthesis among 1-, 2- and 3-year Old Atractylodes Chinensis

10.21203/rs.3.rs-272708/v1 ◽

2021 ◽

Author(s):

Jianhua Zhao ◽

Chengzhen Sun ◽

Shanshan Ma ◽

Jinshuang Zheng ◽

Xin Du ◽

...

Keyword(s):

Rna Sequencing ◽

Transcriptome Analysis ◽

High Performance ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Bioactive Compound ◽

Pcr Analysis ◽

Squalene Epoxidase ◽

Sequencing Analysis ◽

Qrt Pcr

Abstract Background: Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atrctylodin, a sesquiterpenoids. High-performance liquid chromatography (HPLC) analysis demonstrated that atrctylodin was most abundant in 3-year old A. chinensis rhizomes, compared with those from 1-year and 2-year-old plants, however, the molecular mechanisms underlying accumulation of atrctylodin in rhizomes are poorly understood. Results: In this study, we characterized the transcriptomes from 1-, 2, and 3-year old (Y1, Y2, and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 205 and 226 unigenes encoding the enzyme genes in the mevalonate (MVA) and methylerythritol phosphate (MEP) sesquiterpenoid biosynthesis pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven genes key genes encoding factors involved in the sesquiterpene biosynthetic pathway, as well as in pigment, amino acid, hormone, and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson’s correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of samples from different ages revealed 52 genes related to sesquiterpenoids biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3, and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase, squalene-hopene cyclase, squalene epoxidase and dammarenediol II synthase. These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. Conclusion: The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenes in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

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Comparative transcriptome analysis reveals sesquiterpenoid biosynthesis among 1-, 2- and 3-year old Atractylodes chinensis

BMC Plant Biology ◽

10.1186/s12870-021-03131-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jianhua Zhao ◽

Chengzhen Sun ◽

Fengyu Shi ◽

Shanshan Ma ◽

Jinshuang Zheng ◽

...

Keyword(s):

Rna Sequencing ◽

Transcriptome Analysis ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Pcr Analysis ◽

Squalene Epoxidase ◽

Sequencing Analysis ◽

Comparative Transcriptome ◽

Comparative Transcriptome Analysis ◽

Qrt Pcr

Abstract Background Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. Results In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson’s correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. Conclusion The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

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GTR1 and GTR2 transporters differentially regulate tissue‐specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea

Plant Cell & Environment ◽

10.1111/pce.14072 ◽

2021 ◽

Author(s):

Deepti M. Nambiar ◽

Juhi Kumari ◽

Rehna Augustine ◽

Pawan Kumar ◽

Prabodh K. Bajpai ◽

...

Keyword(s):

Brassica Juncea ◽

Oilseed Crop ◽

Tissue Specific ◽

Defence Responses

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Effect of Nitrogen Fertilisation and Inoculation with Bradyrhizobium japonicum on the Fatty Acid Profile of Soybean (Glycine max (L.) Merrill) Seeds

Agronomy ◽

10.3390/agronomy11050941 ◽

2021 ◽

Vol 11 (5) ◽

pp. 941

Author(s):

Ewa Szpunar-Krok ◽

Anna Wondołowska-Grabowska ◽

Dorota Bobrecka-Jamro ◽

Marta Jańczak-Pieniążek ◽

Andrzej Kotecki ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid Composition ◽

Fatty Acid ◽

Acid Composition ◽

Bradyrhizobium Japonicum ◽

Field Experiments ◽

Fatty Acid Content ◽

Soybean Seeds ◽

Oilseed Crop ◽

Soybean Cultivars

Soybean is a valuable protein and oilseed crop ranked among the most significant of the major crops. Field experiments were carried out in 2016–2019 in South-East Poland. The influence of soybean cultivars (Aldana, Annushka), nitrogen fertilizer (0, 30, 60 kg∙ha−1 N) and inoculation with B. japonicum (control, HiStick® Soy, Nitragina) on the content of fatty acids (FA) in soybean seeds was investigated in a three-factorial experiment. This study confirms the genetic determinants of fatty acid composition in soybean seeds and their differential accumulation levels for C16:0, C16:1, C18:1n9, C18:2, C18:3, and C20:0 as well saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids. Increasing the rate from 30 to 60 kg ha−1 N did not produce the expected changes, suggesting the use of only a “starter” rate of 30 kg ha−1 N. Inoculation of soybean seeds with a strain of Bradyrhizobium japonicum (HiStick® Soy, BASF, Littlehampton, UK and Nitragina, Institute of Soil Science and Plant Cultivation–State Research Institute, Puławy, Poland) is recommended as it will cause a decrease in SFA and C16:0 acid levels. This is considered nutritionally beneficial as its contribution to total fatty acids determines the hypercholesterolemic index, and it is the third most accumulated fatty acid in soybean seeds. The interaction of cultivars and inoculation formulation on fatty acid content of soybean seeds was demonstrated. An increase in the value of C16:0 content resulted in a decrease in the accumulation of C18:1, C18:2, and C18:3 acids. The content of each decreased by almost one unit for every 1% increase in C16:0 content. The dominant effect of weather conditions on the FA profile and C18:2n6/C18:3n3 ratio was demonstrated. This suggests a need for further evaluation of the genetic progress of soybean cultivars with respect to fatty acid composition and content under varying habitat conditions.

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Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

Parasites & Vectors ◽

10.1186/s13071-021-04773-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Lenka Ulrychová ◽

Pavel Ostašov ◽

Marta Chanová ◽

Michael Mareš ◽

Martin Horn ◽

...

Keyword(s):

In Situ Hybridization ◽

Schistosoma Mansoni ◽

Rna Sequencing ◽

Serine Proteases ◽

Expression Patterns ◽

High Sensitivity ◽

Host Parasite Interactions ◽

Highly Sensitive ◽

Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

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Transformation and functional verification of Cry5Aa in cotton

Scientific Reports ◽

10.1038/s41598-021-82495-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shihao Zhao ◽

Feng Wang ◽

Qiuping Zhang ◽

Jiayi Zou ◽

Zhangshu Xie ◽

...

Keyword(s):

Resistance Gene ◽

Insect Resistance ◽

Expression Patterns ◽

Instar Larva ◽

Cotton Bollworm ◽

Pcr Analysis ◽

Functional Verification ◽

Study Results ◽

Significant Difference ◽

Transgenic Strains

AbstractMost of the cotton bollworm-resistant genes applied in cotton are more than 20 years and they all belong to Cry1Ab/c family, but the insect-resistant effects of Cry5Aa on cotton were rarely reported. The possible risk of resistance is increasing. The study synthesized a novel bollworm-resistant gene Cry5Aa artificially based on preferences of cotton codon. The new gene was transferred to cotton through the method of pollen tube pathway. The transgenic strains were identified by kanamycin test in field and laboratory PCR analysis. Meanwhile, an insect resistance test was conducted by artificial bollworm feeding with transgenic leaves and GK19 was used as a control in this study. Results showed that rate of positive transgenic strains with kanamycin resistance in the first generation (T1), the second generation (T2) and the third generation (T3) respectively were 7.76%, 73.1% and 95.5%. However, PCR analysis showed that the positive strain rate in T1, T2 and T3 were 2.35%, 55.8% and 94.5%, respectively. The resistant assay of cotton bollworm showed that the mortality rate of the second, third and fourth instar larva feed by the transgenic cotton leaves, were 85.42%, 73.35% and 62.79%, respectively. There was a significant difference between transgenic plant of Cry5Aa and GK19 in insect resistance. Finally, we also conducted the further analysis of gene expression patterns, gene flow and the effect on non-target pest in the study. The results showed that Cry5Aa gene had less environmental impact, and Cry5Aa has been transferred successfully and expressed stably in cotton. Therefore, the novel bollworm resistance gene can partially replace the current insect-resistance gene of Lepidoptera insects.

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Identification, Expression, and Functions of the Somatostatin Gene Family in Spotted Scat (Scatophagus argus)

Genes ◽

10.3390/genes11020194 ◽

2020 ◽

Vol 11 (2) ◽

pp. 194

Author(s):

Peizhe Feng ◽

Changxu Tian ◽

Xinghua Lin ◽

Dongneng Jiang ◽

Hongjuan Shi ◽

...

Keyword(s):

Gene Family ◽

Growth Regulation ◽

Expression Patterns ◽

Open Reading Frames ◽

Pcr Analysis ◽

Sequence Alignments ◽

Scatophagus Argus ◽

Gene Structure And Expression ◽

Liver Fragments

Somatostatins (SSTs) are a family of proteins consisting of structurally diverse polypeptides that play important roles in the growth regulation in vertebrates. In the present study, four somatostatin genes (SST1, SST3, SST5, and SST6) were identified and characterized in the spotted scat (Scatophagus argus). The open reading frames (ORFs) of SST1, SST3, SST5, and SST6 cDNA consist of 372, 384, 321, and 333 bp, respectively, and encode proteins of 123, 127, 106, and 110 amino acids, respectively. Amino acid sequence alignments indicated that all SST genes contained conserved somatostatin signature motifs. Real-time PCR analysis showed that the SST genes were expressed in a tissue specific manner. When liver fragments were cultured in vitro with synthetic peptides (SST1, SST2, or SST6 at 1 μM or 10 μM) for 3 h or 6 h, the expression of insulin-like growth factor 1 and 2 (Igf-1 and Igf-2) in the liver decreased significantly. Treatment with SST5 had no significant effect on Igf-1 and Igf-2 gene expression. This study provides an enhanced understanding of the gene structure and expression patterns of the SST gene family in S. argus. Furthermore, this study provides a foundation for future exploration into the role of SST genes in growth and development.

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