scholarly journals Optimization of a virus-induced gene silencing system and functional elucidation of MaBAM9b in postharvest banana fruits

2020 ◽  
Author(s):  
juhua liu ◽  
Meng Li ◽  
Jing Zhang ◽  
Hongxia Miao ◽  
Jingyi Wang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Fruit Quality ◽  
Gene Function ◽  
Fruit Ripening ◽  
Starch Content ◽  
Soluble Sugar ◽  
Starch Degradation ◽  
Banana Fruit ◽  
Virus Induced Gene Silencing ◽  
Starch Conversion

Abstract Background: Banana is a typical starch conversion fruit, and fruit ripening involves a process of fruit quality formation. To improve the nutritional value of banana, it is necessary to understand the genetic basis of the metabolic pathways that operate during fruit ripening processes. MaBAM9b is a key enzyme gene that might play an important role in starch degradation during the banana fruit ripening process. The identification of gene function by stable genetic transformation is time- and energy-consuming. Thus, developing an efficient and rapid method for functional identification is imperative. Virus-Induced Gene Silencing (VIGS) is a reverse-genetics method based on RNA-mediated antiviral plant defense that has been used to rapidly identify gene function in plants. Results: In this report, 0.5% iodine-potassium-iodide (I2-KI) staining for 150 s determined that a 1:3 ratio of TRV1: TRV2-MaBAM9b cultivated at an optical density of 600 nm (OD600) 0.8 at 30 mmHg for 30 sec and kept on Murashige & Skoog (MS) media for 5 d produced the best silencing results. Under these conditions, the total starch content was greatly increased, while the β-amylase activity, soluble sugar contents, and the expression of endogenous MaBAM9b were greatly decreased. Conclusions: The developed system is particularly useful for studying genes and networks for starch conversion in fruit, which alone would not produce a visual phenotype. This system will provide a platform for banana functional genomics and for banana fruit quality improvement.

scholarly journals Optimization of a virus-induced gene silencing system and functional elucidation of MaBAM9b in postharvest banana fruits

2020 ◽  
Author(s):  
juhua liu ◽  
Meng Li ◽  
Jing Zhang ◽  
Hongxia Miao ◽  
Jingyi Wang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Reverse Genetics ◽  
Starch Content ◽  
Sugar Content ◽  
Soluble Sugar ◽  
Starch Degradation ◽  
Virus Induced Gene Silencing ◽  
Functional Identification ◽  
Exact Function ◽  
Starch Conversion

Abstract Background: The genetic basis of metabolic pathways that operate during fruit ripening needs to be understood before the nutritional value of the banana can be improved. The banana is a typical starch conversion fruit, and β-amylase is a key enzyme that may play an important role in starch degradation during the ripening process. Musa acuminata β-amylase 9b (MaBAM9b) is closely related to starch degradation. However, its exact function in starch degradation has not been demonstrated in banana. Stable genetic transformation to identify gene function is time- and energy-consuming. Thus, an efficient and rapid method is needed for functional identification. Virus-induced gene silencing (VIGS) is a reverse-genetics method based on RNA-mediated antiviral plant defense that has been used to rapidly identify gene functions in plants. The aim of this study is to optimize a VIGS system and functional elucidation of MaBAM9b in postharvest banana fruits. Results: Using 0.5% iodine-potassium-iodide (I2-KI) staining for 150 s, we found that 1:3 TRV1:TRV2-MaBAM9b cultivated at 30 mmHg for 30 s to an optical density (OD) of 0.8 at 600 nm, and kept on Murashige & Skoog (MS) media for 5 days produced the best silencing results. Under these conditions, the total starch content was greatly increased, whereas the β-amylase activity, soluble sugar content, and expression of endogenous MaBAM9b greatly decreased. Conclusions: The system described here is particularly useful for studying genes and networks involved in starch conversion in fruit, which alone would not produce a visual phenotype. This system will provide a platform for functional genomics and fruit quality improvement in the banana.

scholarly journals Applications of virus-induced gene silencing for identification of gene function in fruit

2021 ◽  
Vol 5 ◽  
Author(s):  
Gangshuai Liu ◽  
Hongli Li ◽  
Daqi Fu
Keyword(s):  
Gene Silencing ◽  
Fruit Quality ◽  
Gene Function ◽  
Biological Process ◽  
Virus Induced Gene Silencing ◽  
Functional Identification ◽  
Advantages And Disadvantages ◽  
Research Fields ◽  
Complex Biological Process

Abstract With the development of bioinformatics, it is easy to obtain information and data about thousands of genes, but the determination of the functions of these genes depends on methods for rapid and effective functional identification. Virus-induced gene silencing (VIGS) is a mature method of gene functional identification developed over the last 20 years, which has been widely used in many research fields involving many species. Fruit quality formation is a complex biological process, which is closely related to ripening. Here, we review the progress and contribution of VIGS to our understanding of fruit biology and its advantages and disadvantages in determining gene function.

scholarly journals The Involvement of the Banana F-Box Protein MaEBF1 in Regulating Chilling-Inhibited Starch Degradation through Interaction with a MaNAC67-Like Protein

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 552 ◽  
Author(s):  
Song ◽  
Qin ◽  
Zheng ◽  
Ding ◽  
Chen ◽  
...  
Keyword(s):  
Starch Content ◽  
Sugar Content ◽  
Soluble Sugar ◽  
Chilling Injury ◽  
Storage Conditions ◽  
Starch Degradation ◽  
Banana Fruit ◽  
Ethylene Signaling ◽  
Low Temperature Storage ◽  
Vegetables And Fruits

Low-temperature storage is a common strategy for preserving and transporting vegetables and fruits. However, many fruits are hypersensitive to chilling injury, including bananas. In the present study, storage conditions of 11 °C delayed the ripening of Fenjiao (Musa ABB Pisang Awak) banana, and the pulp could be softened after ethephon treatment. Storage conditions of 7 °C prevented fruit from fully softening, and fruit contained a significantly higher starch content and lower soluble sugar content. MaEBF1, a critical gene component in the ethylene signaling pathway, was repressed during ripening after fruit had been stored for 12 days at 7 °C. The expression of a series of starch degradation-related genes and a MaNAC67-like gene were also severely repressed. Both MaEBF1 and MaNAC67-like genes were ethylene-inducible and localized in the nucleus. MaNAC67-like protein was able to physically bind to the promoter of genes associated with starch degradation, including MaBAM6, MaSEX4, and MaMEX1. Yeast two-hybrid, GST-pull down, and BiFC assays showed that MaEBF1 interacted with the MaNAC67-like protein, and their interaction further activated the promoters of MaBAM6 and MaSEX4. The current study indicates that MaNAC67-like is a direct regulator of starch degradation and potential for involvement in regulating chilling-inhibited starch degradation by interacting with the ethylene signaling components in banana fruit. The present work paves the way for further functional analysis of MaEBF1 and MaNAC67-like in banana, which will be useful for understanding the regulation of banana starch metabolism and fruit ripening.

scholarly journals A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Decai Tuo ◽  
Peng Zhou ◽  
Pu Yan ◽  
Hongguang Cui ◽  
Yang Liu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Viral Vector ◽  
Function Analysis ◽  
Systemic Infection ◽  
Cdna Clones ◽  
Virus Induced Gene Silencing ◽  
Efficient Gene ◽  
Gene Function Analysis

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Study on some of the physiological and biochemical indexes under the age of development of banana fruit (Musa paradisiaca L.) grown in Thanh Tri, Hanoi

2021 ◽  
Vol 66 (1) ◽  
pp. 87-95
Author(s):  
Trong Le Van ◽  
Khanh Nguyen Nhu
Keyword(s):  
Fruit Ripening ◽  
Starch Content ◽  
Sugar Content ◽  
Maximum Size ◽  
Research Process ◽  
Scientific Basis ◽  
Carotenoid Content ◽  
Banana Fruit ◽  
Fruit Formation ◽  
Physiological And Biochemical

Research to determine the ripening time of the fruit is the scientific basis for better harvesting and preservation. Physiological and biochemical methods were used to analyze the changes of some indicators according to the growth and development of banana fruit grown in Thanh Liet commune, Thanh Tri district, Hanoi from the time of its formation until the fruit ripening. The results showed that the banana reached the maximum size at 16 weeks old, at this time the peel was yellow due to the decrease in chlorophyll and increased carotenoid content. The content of vitamin C and total organic acid content reached their maximum when the fruit at 12 weeks old, then decreased gradually. Starch content increased to 14 weeks old, then decreased. Reduced sugar content increased gradually to 16 weeks old and then decreased. Protein content decreased gradually from fruit formation until fruit ripening, lipid content increased gradually to 15 weeks old, then decreased. Through the research process, we have determined that the physiological ripe time of banana fruit was 16 weeks old, this is the time when the fruit stops growing and accumulates many nutrients.

scholarly journals An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits

2021 ◽  
Vol 12 ◽  
Author(s):  
Yifan Wang ◽  
Ning Huang ◽  
Niu Ye ◽  
Lingyu Qiu ◽  
Yadong Li ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Function Analysis ◽  
Juglans Regia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tobacco Rattle Virus ◽  
Post Inoculation ◽  
Virus Induced Gene Silencing ◽  
Gene Function Analysis

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

scholarly journals Rose Phytoene Desaturase Gene Silencing by Apple Latent Spherical Virus Vectors

HortScience ◽  
2012 ◽  
Vol 47 (9) ◽  
pp. 1278-1282 ◽  
Author(s):  
Hiroaki Ito ◽  
Masaki Ochiai ◽  
Hiroaki Kato ◽  
Katsuhiro Shiratake ◽  
Daigo Takemoto ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Molecular Breeding ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Desaturase Gene ◽  
Apple Latent Spherical Virus ◽  
The Rose ◽  
Highly Uniform ◽  
Secondary Shoot

We have succeeded in establishing a virus-induced gene silencing (VIGS) of rose using Apple latent spherical virus (ALSV) vectors. An ALSV infection on rose did not cause any symptoms like those observed on other plant species and grew healthy. We have cloned and sequenced the phytoene desaturase (PDS) gene in wild rose, then used its fragment for silencing the rose internal PDS gene. The silencing phenotypes such as the highly uniform photo-bleached phenotype with PDS inhibitions were observed on the upper leaves of primary shoots and on a secondary shoot of R. rugosa for more than 5 months. ALSV vectors seemed useful for analyzing gene function and for the molecular breeding of rose.

scholarly journals A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

2019 ◽  
Author(s):  
Mei Liu ◽  
Zhiling Liang ◽  
Miguel A. Aranda ◽  
Ni Hong ◽  
Liming Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Nicotiana Benthamiana ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Economic Significance ◽  
Potential Alternative ◽  
Low Efficiency

AbstractCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiencies. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits. Here, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69-300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged. These results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function in cucurbits.One sentence summaryA CGMMV-based vector enables gene function studies in cucurbits, an extremely low efficiency species for genetic transformation.

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