Optogenetic Rescue of a Patterning Mutant

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应用体细胞基因转移技术建立的遗传性极度高血脂小鼠模型

目的　利用体细胞脂蛋白脂酶 (lipoproteinlipase ,LPL)有益变异体基因转移方法 ,救治原本在出生后两天内全部死亡的LPL基因敲除纯合子小鼠。方法　以腺病毒为载体 ,在初生小鼠肌肉内表达LPL基因有益突变体 ,观察救治存活后成年动物的表型变化。结果　本法救治纯合子小鼠的成功率达到 75 % ,大大高于以野生型LPL基因救治的国外研究。存活的成年纯合子小鼠表现为极度高脂血症。结论　利用LPL基因突变体进行体细胞基因转移可成功救治LPL基因敲除纯合子小鼠 ,并建立了极度高脂血症动物模型。     

Somatic Embryogenesis in Cultured Carrot Cells

Somatic embryogenesis in cultured plant cells is an ideal system for investigating the whole process of differentiation and development from single cells to whole plants, and especially the molecular mechanism of expression of totipotency. This review reports recent progress the studies on somatic embryogenesis.     

Rescue of a Dominant Mutant With RNA Interference

Maize Mucronate1 is a dominant floury mutant based on a misfolded 16-kDa γ-zein protein. To prove its function, we applied RNA interference (RNAi) as a dominant suppressor of the mutant seed phenotype. A γ-zein RNAi transgene was able to rescue the mutation and restore normal seed phenotype. RNA interference prevents gene expression. In most cases, this is used to study gene function by creating a new phenotype. Here, we use it for the opposite purpose. We use it to reverse the creation of a mutant phenotype by restoring the normal phenotype. In the case of the maize Mucronate1 (Mc1) phenotype, interaction of a misfolded protein with other proteins is believed to be the basis for the Mc1 phenotype. If no misfolded protein is present, we can reverse the mutant to the normal phenotype. One can envision using this approach to study complex traits and in gene therapy.TRANSLUCENT or vitreous maize kernels are harder and able to sustain stronger mechanical strength during harvesting, transportation, and storage. There is a direct link between a vitreous seed phenotype and the type of storage proteins in the seed, collectively called zeins in maize. Zeins, encoded by a multigene family, constitute >60% of all maize seed proteins. They are classified into four groups (α-, β-, γ-, and δ-zein) on the basis of their structures (Esen 1987). Zeins are specifically synthesized in the endosperm ∼10 days after pollination (DAP) and deposited into protein bodies (Wolf et al. 1967; Burr and Burr 1976; Lending and Larkins 1992). Irregularly shaped protein bodies are found in floury or opaque kernel phenotypes (Coleman et al. 1997; Kim et al. 2004, 2006; Wu et al. 2010; Wu and Messing 2010). The terms “floury” and “opaque” were originally created on the basis of the genetic behaviors of the mutant allele causing the soft kernel texture. The floury mutants behave as semidominant or dominant mutants, as floury1 and floury2 do, while the opaque mutants are recessive, as opaque1 and opaque2 are (Hayes and East 1915; Lindstrom 1923; Emerson et al. 1935; Maize Genetics Cooperation 1939). Similar to floury2 with a single mutation in the signal peptide of a 22-kDa α-zein resulting in an unprocessed protein (Coleman et al. 1995), De*-B30 produces an unprocessed 19-kDa α-zein (Kim et al. 2004). It was hypothesized that the two mutant proteins with an unprocessed signal peptide are misfolded and docked in the membranes of the rough endoplasmic reticulum (RER), blocking the deposition of other zein proteins (Coleman et al. 1995; Kim et al. 2004). In Mucronate1 (Mc1), a 38-bp deletion in the C terminus of the 16-kDa γ-zein (γ16-zein) gene resulted in a frameshift and a protein with a different amino-acid tail. This modified 16-kDa γ-zein (Δγ16-zein) has altered solubility properties, which would explain the formation of irregular protein bodies. Because De*-B30 and Mc1 are semidominant and dominant, respectively, they belong to the floury mutant class.The γ-zein genes (γ27-zein and γ16-zein) are homologous copies because maize underwent allotetraploidization and both gene copies have been retained during diploidization (Xu and Messing 2008). The two γ-zeins and the 15-kDa β-zein have a redundant function in stabilizing protein-body formation (Wu and Messing 2010). Knockdown of both γ-zeins with a single RNA interference (RNAi) construct conditioned only partial opacity in the crown, the top of the kernel, as opposed to the remainder or gown area of the kernel. Consistent with its light kernel phenotype, protein bodies in such a γ-zein RNAi (γRNAi) mutant exhibited a slight alteration in morphology. This phenotype is clearly distinguishable from the Mc1 phenotype, which is far more severe. Therefore, if Mc1 is caused by a misfolded chimeric 16-kDa γ-zein, preventing its expression should restore normal kernel phenotype. Indeed, a simple cross of Mc1 with a maize line carrying the γRNAi transgene produced a non-floury phenotype, providing an example of RNAi as a dominant suppressor of a dominant phenotype and as a general tool in marker rescue.

Analysis of the progeny from the cross of Mc1 and γRNAi mutants:

Mc1 seeds (Stock ID U840I) were requested from the Maize Genetics Cooperation Stock Center. The γRNAi transgenic lines have been reported in previous work (Wu et al. 2010; Wu and Messing 2010). Twelve progeny kernels from the cross of the Mc1 mutant [homozygous for the dominant-negative mutant 16-kDa γ-zein alleles (Δγ16/Δγ16) and heterozygous for the γRNAi line (γRNAi/+)] were dissected at 18 DAP for segregation and mRNA accumulation analyses. For each kernel, the embryo and endosperm were separated for DNA and RNA extraction, respectively. As shown in Figure 1A, five and seven kernels were positive and negative for the amplification of the γRNAi gene with a specific primer set, exemplifying a 1:1 segregation of the γRNAi gene.Open in a separate windowFigure 1.—Segregation analysis of the accumulations of mRNAs and proteins from the cross of the Mc1 mutant and the γRNAi line by RT–PCR and SDS–PAGE. (A) γRNAi gene segregation from progeny (Δγ16/Δγ16 x γRNAi/+) by PCR amplification with a specific primer set (GFPF, ACAACCACTACCTGAGCAC and T35SHindIII, ATTAAGCTTTGCAGGTCACTGGATTTTGG). Kernels 3, 8, 9, 10, and 12 are positive for the γRNAi gene and the rest of them are negative. M, DNA markers from top to bottom band are 3, 2, 1.5, 1.4, and 1 kb. (B) RT–PCR analysis of mRNA accumulation from the normal γ16 and mutant Δγ16 alleles in the endosperms with the genotypes corresponding to the embryos analyzed above. Total RNA was extracted by using TRIzol reagent (Invitrogen). Two micrograms of RNA was digested with DNase I (Invitrogen) and then reverse-transcribed. Twenty-five nanograms of cDNA from each of the twelve endosperms was applied for PCR (25 cycles of 30 sec, 94 °C; 30 sec, 58 °C; and 1 min, 72 °C). A specific primer set (γ16F, ATGAAGGTGCTGATCGTTGC and γ16R, TCAGTAGTAGACACCGCCG) was designed for amplification of the full-length γ16-zein coding sequence (552 bp). The lower band (514 bp) from the mutant Δγ16 allele is 38 bp shorter than that from the normal allele (552 bp). Kernels 3, 8, 9, 10, and 12 with the γRNAi gene accumulated significantly less mRNA compared to those without the γRNAi gene (kernels 1, 2, 4, 5, 6, 7, and 11). BA, hybrid of B × A lines. M, DNA markers from top to bottom are 1 kb, 750 bp, and 500 bp. (C) Profile of zein accumulations of 20 kernels from the progeny as described in the text. The zein extraction method has been described elsewhere (Wu et al. 2009). The Δγ16-zein from Mc1 was not extracted by traditional total-zein extraction protocol (70% ethanol and 2% 2-mercaptoethanol). The γ27- and γ16-zeins were knocked down to a nondetectable level in kernels 1, 2, 3, 5, 7, 10, 12, 13, 16, and 20. In γRNAi-gene segregating progeny (kernels 4, 6, 8, 9, 11, 14, 15, 17, 18, and 19), the γ16-zein from the normal γ16 allele is marked by arrowheads. Protein loaded in each lane was equal to 500 μg fresh endosperm at 18 DAP. The size for each band is indicated by the numbers in the “kDa” columns. BA, hybrid of B × A lines; 1–20, kernels from the progeny described above; M, protein markers from top to bottom are 50, 25, 20, and 15 kDa.Due to the 38-bp deletion in the C terminus of the coding region, the Δγ16 allele is shorter than the normal one (Figure 1B). Therefore, most of Δγ16-zein was in the non-zein fraction. In progeny endosperms of another 20 kernels from the same cross described above segregating for the γRNAi gene, two types of γ16-zeins were synthesized: the normal γ16-zein in the ethanol-soluble zein fraction and the Δγ16-zein in the non-zein fraction. In progeny inheriting the γRNAi gene, the γ27- and γ16-zeins were reduced to nondetectable levels (Figure 1C). Although the Δγ16-zein is not in the ethanol-soluble zein fraction, the level of normal γ16-zein is a good indicator of the accumulation of the Δγ16-zein.

Rescue of protein-body morphologies in the Mc1 mutant:

Regular protein bodies are round with distinct membrane boundaries (Figure 2A) and 1–2 μm in diameter at maturity. In homozygous and heterozygous Mc1 mutants (Δγ16/Δγ16 and Δγ16/+), protein bodies were irregularly shaped, some without discrete boundaries (Figure 2, C and D), which is quite different from the absence of normal γ27- or γ16-zeins in maize endosperm (Figure 2B). Indeed, protein bodies of the Mc1 mutant, blocked in the accumulation of Δγ16-zein, showed morphologies with no discernible difference from those in the γRNAi/+ line (Figure 2, B and E).Open in a separate windowFigure 2.—Transmission electron micrographs of protein bodies. The method has been described elsewhere (Wu and Messing 2010). (A) Nontransgenic BA. (B) γRNAi transgenic line (γRNAi/+). (C) Mc1 (Δγ16/Δγ16). (D) Cross of Mc1 mutant and nontransgenic hybrid of B × A lines (Δγ16/+). (E) Cross of Mc1 mutant (Δγ16/Δγ16) and heterologous γRNAi transgenic line (γRNAi/+). PB, protein body; RER, rough endoplasmic reticulum; CW, cell wall; Mt, mitochondria; SG, starch granule. Bars, 500 nm.

Recovery of floury phenotype in progeny:

On the basis of these observations, it is reasoned that irregularly shaped protein bodies (Figure 2, C and D) in the Mc1 mutant cause the floury phenotype (Figure 3, A and B). Because knockdown of γ-zeins caused opacity only in the crown area (Figure 3C), one could envision that once the irregular protein bodies are restored, the kernel would become vitreous in the gown area of the kernel. Indeed, the progeny ear from the cross of Δγ16/Δγ16 and γRNAi/+ showed a 1:1 ratio of floury and vitreous kernels (Figure 3, D and F), and all kernels were vitreous when the Mc1 mutant was pollinated by a homozygous γRNAi line (Figure 3E).Open in a separate windowFigure 3.—Segregation of vitreous and floury kernels from a progeny ear. (A) Mc1 mutant with Δγ16/Δγ16 genotype. (B) The cross of the Mc1 mutant and the nontransgenic hybrid of B × A lines, showing floury phenotype as in A. (C) γRNAi transgenic line with partial opacity only in the crown area. (D) The cross of the Mc1 mutant (Δγ16/Δγ16) and the heterologous γRNAi transgenic line (γRNAi/+), showing a 1:1 ratio of vitreous and floury kernels. A row in the ear is marked with arrowheads and crosses to indicate vitreous and floury gowns of kernels. (E) Cross of the Mc1 mutant (Δγ16/Δγ16) and the γRNAi homozygous transgenic line (γRNAi/γRNAi), showing all vitreous kernels. (F) Truncated kernel phenotype. (Top) Mc1, cross of Mc1 × BA, and γRNAi transgenic line. (Bottom) Three vitreous and floury kernels from D.

Conclusions:

RNAi can be used to rescue mutations that are dominant negative with a single cross, providing a useful tool in genetic analysis, plant breeding, and potentially in gene therapy in general.     

水稻纯合胚致死突变研究

以ＥＭＳ的处理并结合组织培养技术成功地获得胚致死突变的纯合再生植株。该植株生长发育正常,除种子无发芽能力外,纯合突变体的一切性状均与亲本品种表现一致。观察到胚败育的各种表现：（１）仅具有一个球形胚。（２）完全没有胚器的分化。（３）仅具胚根的分化而无胚芽的分化。（４）胚芽分化不完全。（５）胚芽与胚根之间没有输导组织相连接或者输导组织发育不完全等等。（纯合突变体×正常品种）杂种当代的种子（Ｆ１）发芽正常,而由Ｆ１及Ｒ１植株上所产生的种子（Ｆ２及Ｒ２）约有３／４具发芽能力,而１／４无发芽能力。统计分析的结果表明胚致死突变受隐性单基因控制。据我们所知,获得胚致死突变纯合体的成熟植株,本研究乃是首例报告,至少在水稻上是如此。在以利用无融合生殖之固定杂种优势的“一系法”杂交水稻生产的设想中,胚致死突变可作为胚乳的提供者而加以利用。     

Precipitated Proteins Improve the Production of Carrot Somatic Embryos

Extracellular compounds isolated from embryogenic carrot cell suspension cultures increase, by 1.5 to 6-fold, end-stage embryo production when added back to carrot cultures initiating embryogenesis. The causative factors related to the enhancement of embryo production are most likely to be extracellular, high molecular weight proteins found in the embryo-free medium (EFM) after somatic embryos have been formed. The addition of heat-treated EFM to fresh cultures did not result in enhancing effects on the production of end-stage embryos. However, the addition of compounds precipitated from EFM, by high concentrations of salt, accelerated by four days the formation of comparable amounts of end-stage embryos and surpassed total end-stage embryo levels by a factor of 4-6, dependent on the precipitate dose. These results suggest that heat-labile polypeptide molecules may be responsible for growth factor-like effects during somatic embryogenesis.     

黑杨胚抢救技术体系优化

采用组织培养技术进行黑杨胚抢救和比较不同黑杨派树种杂交组合以及不同胚抢救时间的结果表明黑杨胚抢救的最佳时间范围为授粉后30-35d。已长有3支以上须根的胚培苗进行移栽时保持的温度为20-25℃,湿度为70％左右,3d可成活,1周即可完全适应外界环境。     

Differential Accumulation of Biotin Enzymes during Carrot Somatic Embryogenesis

The activities of four biotin enzymes, acetyl-coenzyme A (CoA) carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase, and the accumulation of six biotin-containing polypeptides were determined during development of somatic embryos of carrot (Daucus carota). Acetyl-CoA carboxylase activity increased more than sevenfold, whereas the activities of 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase were relatively unaltered. An increase also occurred in the accumulation of three of the biotin-containing polypeptides (molecular masses of 220, 62, and 34 kilodaltons). Of these, the most dramatic change was in the accumulation of the 62-kilodalton biotin-containing polypeptide, which increased by at least 50-fold as embryogenic cell clusters developed into torpedo embryos.     

Using GFP as a Scorable Marker in Walnut Somatic Embryo Transformation

Somatic embryogenesis is the foundation of genetic transformationin several economically important tree species. In the Juglansregia L. (Persian walnut) somatic embryogenesis-based transformationsystem, a major limiting factor is the selection of non-chimerictransgenic embryos in tissue culture. We transformed Persianwalnut somatic embryos with the S65T synthetic green fluorescentprotein (GFP) gene in order to assess the effect of this visualmarker gene on embryo viability and the selection of transgenicembryos. Following a 10 d period of transient GFP expressionin all inoculated embryos, stable fluorescent sectors were apparentin several embryos, allowing efficient and rapid visual selectionof primary transgenic embryos. Two chimeric embryos were selected40 d after transformation, and these two embryos gave rise to13 stable transgenic embryo lines and 44 whole plants. GFP-expressingwalnut plants and embryos developed normally and transformationwas verified by GUS analysis. Our analysis suggests that theuse of GFP as a selectable marker can significantly reduce labour,cost, and time in the walnut somatic embryogenesis-based transformationsystem. Copyright 2000 Annals of Botany Company Juglans regia, Persian walnut, somatic embryogenesis, transformation, GFP, scorable marker, in-culture selection efficiency, ß-glucuronidase, fluorescence microscopy     

The Role of Calcium and Calmodulin in Carrot Somatic Embryogenesis

The role of Ca²⁺ and calmodulin in carrot somatic embryo formationwas examined. Embryogenic cell clumps were induced to form embryosin medium containing 0–3 mM Ca²⁺. Embryo formation wasnot affected until the concentration of Ca²⁺ was lower than200 µM and after this threshold was reached the percentof embryo formation decreased with lower Ca²⁺ concentrations.Treatment of developing embryos with either verapamil or nifedipine,Ca²⁺ -channel blockers, or the Ca²⁺ ionophore A23187 [GenBank] , inhibitedembryo formation. These results suggest that exogenous Ca²⁺or the maintenance of Ca²⁺ gradients are required for properembryo development. Analysis of membrane-associated Ca²⁺ andtotal membrane distribution using the fluorescent dyes chlorotetracyclineand N-phenyl-1-napthylamine, respectively, indicated changesin the distribution of membranes during embryogenesis withoutany significant alterations in the concentration of Ca²⁺ associatedwith the membranes. In heart- and torpedo-stage embryos, calmodulin-Ca²⁺complexes were localized in regions containing the developingmeristems of both the cotyledon tips and rhizoid regions whiletotal calmodulin protein appeared to be more uniformly distributed.Calmodulin mRNA levels increased slightly when cell clumps wereinduced to form embryos. (Received July 7, 1993; Accepted September 27, 1993)     

胡萝卜体细胞胚胎发生中的细胞组织化学和蛋白质组成变化

研究胡萝卜体细胞胚不同发育阶段的细胞组织化学和蛋白质组成变化的结果表明:胚性愈伤组织主要源自维管束周围的细胞.球胚形成前期,淀粉粒和糊粉粒极性分布已很明显.子叶胚期,芽开始分化,有大量糊粉粒累积.在体细胞胚发育过程中,淀粉粒在胚性愈伤组织形成初期和球胚后期、糊粉粒在胚性愈伤组织形成后期和球胚期各有两次累积高峰.     

Oxidative Metabolism of a Pigmented Mutant of Rhizobium meliloti and its Revertants

Non-infective pigmented mutants of Rhizobium meliloti have been obtained by u.v. irradiation. The majority of colourless revertants of a pigmented mutant obtained by u.v. irradiation nodulated the host plant indicating that pigmentation in rhizobium and ability to nodulate are genetically linked and may have some pleiotrophic effect. All the nodulating revertants and the parent strain oxidized pentoses, hexoses, dihexoses, tricarboxylic acid (TCA) cycle intermediates and related compounds at a higher rate than the non-nodulating revertants and pigmented mutant. Significant differences in the oxidation of pentoses and TCA cycle intermediates in nodulating and non-nodulating revertants indicate that these two energy yielding pathways are operating less efficiently in non-nodulating revertants and suggests that the working of these two pathways may be essential for the nodulation process.     

Isolation of Carrot Argonaute1 from Subtractive Somatic Embryogenesis cDNA Library

Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.     

Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended [beta]-sheet conformation and had a reduced proportion of [alpha]-helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a [beta]-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.     

胡萝卜人工种子包衣材料的筛选

以胡萝卜SK4-316种子苗下胚轴为外植体诱导培养的体细胞胚,进行人工种子的制作。结果表明,海藻酸钠包埋体系制作的人工种子萌发率较稳定,PEG体系的萌发率高于前者,但萌发后成苗率较低。以MS液体培养基作包埋培养基,4%海藻酸钠、2% CaCl₂·2H₂O、0.1 mg/L GA₃、0.2%活性炭组成的海藻酸钠复合包衣剂制作的人工种子,在1/16MS培养基、24℃下培养萌发率最高,可达97.02%。     

‘SK4—316’胡萝卜体胚的诱导和培养

以'SK4-316'胡萝卜无菌苗的下胚轴为外植体,研究不同培养基配方和培养条件对愈伤组织诱导、体细胞胚间接发生及其同步化培养的影响,以及不同脱分化时间、脱分化培养基及外植体续存时间对体细胞胚直接发生的诱导及其培养的影响.结果表明:含3%蔗糖、0.8%琼脂的1/2MS + 2,4-D 2.5 mg/L + 6-BA(或KT)0.5 mg/L + CH 300 mg/L是诱导愈伤组织的良好培养基;1/2MS + 2,4-D 1.25 mg/L + KT 0.25 mg/L + 6-BA 0.25 mg/L(含3%蔗糖)适于愈伤组织分化并诱导体胚发生,0.02% ABA对体胚的诱导有促进作用,0.06% ABA或15% PEG能促进体胚成熟;外植体在MS + 2,4-D 1.0 mg/L固体培养基上脱分化培养48 h,再转入MS + CH 300 g/L液体培养基中可诱导体胚直接发生,但随着外植体续存于诱导培养基中时间的延长,体胚发生变异的几率也渐增.     

党参的体细胞胚发生及不同发育阶段几种同工酶的分析

以党参为材料,在附加0.1 mg·L-1 2,4-D、0.3 mg·L-1 6-BA和3%蔗糖的MS培养基上获得大量发育良好的体细胞胚. 附加6-BA可以使胚性愈伤组织进行芽的分化,而添加0.1%活性炭后仅有根的分化.利用梯度凝胶电泳进行同工酶的研究表明在胚性愈伤组织和体细胞胚之间,酶谱差异比较大;而在不同发育时期的体细胞胚之间,差异较小.并讨论了同工酶酶谱和酶活性变化与体细胞胚发生、发育的关系.