The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV(white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the p...The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV(white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifesta-tions of hemocytes were similar among moribund shrimps infected via injection,feeding and by nature. Firstly,the total hemocyte counts(THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly,necrotic,broken and dis-integrated cells were often observed,and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly,necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared,whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV,and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.展开更多
Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the c...Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.展开更多
In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbec...In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbeck. Observations of the effect of PGRs were carried out using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D),1-naphthalene acetic acid (NAA) and combinations of 2,4-D and NAA using different basal media. This study found Citrus sinensis (L.) Osbeck exhibited a high frequency of callus induction on MS medium supplemented with 3 mg/L 2,4-D and callus induction frequency was 86.7% ± 3.4% whereas N6 and B5 showed lower callus induction frequency of 83.3% ± 8.8% and 82.2% ± 1.9% respectively compared to that of MS media with supplementation of the same hormone. Among the induced calli, the morphological analysis showed only 40% - 50% was embryogenic calli. Regeneration of plantlets from calli was done using different concentrations and combinations of auxin and cytokinin. The study showed that 3 mg/L 6-benzylaminopurine (BAP) supplemented medium has the maximum potential to promote regeneration of Citrus sinensis (L.) Osbeck from embryogenic calli with the frequency of 89.3% ± 8.8% but no regeneration occurred from the non-embryogenic calli. The regenerated plantlets were rooted on MS medium with supplementation of 5 mg/l NAA. These observations in Citrus sinensis (L.) Osbeck regeneration will be helpful for genetic improvement with desired traits.展开更多
The developmental types of secretory cavities in Citrus remain unclear and whether or not programmed cell death is involved in the developmental process of secretory cavities remains an enigma.Regarding cavity formati...The developmental types of secretory cavities in Citrus remain unclear and whether or not programmed cell death is involved in the developmental process of secretory cavities remains an enigma.Regarding cavity formation in Citrus sinensis fruits,this work uncovered novel evidence to delineate secretory cavity formation in schizolysigeny,supporting the possibility of utilizing secretory cavities as a new cell biology model for investigating the molecular mechanism of plant programmed cell death.展开更多
Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor....Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.展开更多
文摘The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV(white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifesta-tions of hemocytes were similar among moribund shrimps infected via injection,feeding and by nature. Firstly,the total hemocyte counts(THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly,necrotic,broken and dis-integrated cells were often observed,and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly,necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared,whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV,and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.
文摘Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.
文摘In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbeck. Observations of the effect of PGRs were carried out using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D),1-naphthalene acetic acid (NAA) and combinations of 2,4-D and NAA using different basal media. This study found Citrus sinensis (L.) Osbeck exhibited a high frequency of callus induction on MS medium supplemented with 3 mg/L 2,4-D and callus induction frequency was 86.7% ± 3.4% whereas N6 and B5 showed lower callus induction frequency of 83.3% ± 8.8% and 82.2% ± 1.9% respectively compared to that of MS media with supplementation of the same hormone. Among the induced calli, the morphological analysis showed only 40% - 50% was embryogenic calli. Regeneration of plantlets from calli was done using different concentrations and combinations of auxin and cytokinin. The study showed that 3 mg/L 6-benzylaminopurine (BAP) supplemented medium has the maximum potential to promote regeneration of Citrus sinensis (L.) Osbeck from embryogenic calli with the frequency of 89.3% ± 8.8% but no regeneration occurred from the non-embryogenic calli. The regenerated plantlets were rooted on MS medium with supplementation of 5 mg/l NAA. These observations in Citrus sinensis (L.) Osbeck regeneration will be helpful for genetic improvement with desired traits.
基金supported by the National Natural Science Foundation of China(30670119)
文摘The developmental types of secretory cavities in Citrus remain unclear and whether or not programmed cell death is involved in the developmental process of secretory cavities remains an enigma.Regarding cavity formation in Citrus sinensis fruits,this work uncovered novel evidence to delineate secretory cavity formation in schizolysigeny,supporting the possibility of utilizing secretory cavities as a new cell biology model for investigating the molecular mechanism of plant programmed cell death.
基金supported by the Fundamental Research Funds for the Central Universities (Grant No. XDJK 2018B016)the National Natural Sciences Foundation of China (Grant No. 31972393)+1 种基金he earmarked fund for China Agriculture Research System (Grant No. CARS-26)the Natural Science Foundation of Chongqing (Grant No. cstc2020jcyj-msxmX1064)。
文摘Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.
